Replicative minicircle vectors with improved expression

ABSTRACT

The present invention relates to the production and use of covalently closed circular (ccc) recombinant DNA molecules such as plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof, and more particularly to vector modifications that improve expression of said DNA molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.14/432,693, filed on Mar. 31, 2015 entitled “Replicative MinicircleVectors with Improved Expressions” which is a 371 U.S. National Phase ofInternational Application PCT/US2013/000259, filed Nov. 18, 2013, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/796,765, entitled “Replicative Minicircle vectors with improvedexpression” filed Nov. 19, 2012, the entire contents of which areincorporated herein by reference.

This application also claims priority to International ApplicationPCT/US13/00067, entitled “Replicative Minicircle vectors with improvedexpression” filed Mar. 14, 2013, and International Patent ApplicationPCT/US13/00068, entitled “DNA plasmids with improved expression” filedMar. 14, 2013, the entire contents of which are incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported in part with government support under GrantNo. R44GM080768, awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

INCORPORATION-BY-REFERENCE

The accompanying Sequence Listing incorporates by reference the materialin the ASCII text file identified by the file name: Seq_listing.txt,creation date: Apr. 4, 2016, size: 95 kilobytes.

FIELD OF THE INVENTION

The present invention relates to a family of eukaryotic expressionplasmids useful for gene therapy, obtaining improved geneticimmunization, natural interferon production, and more particularly, forimproving the expression of plasmid encoded antigens or therapeuticgenes.

Such recombinant DNA molecules are useful in biotechnology, transgenicorganisms, gene therapy, therapeutic vaccination, agriculture and DNAvaccines.

BACKGROUND OF THE INVENTION

E. coli plasmids have long been an important source of recombinant DNAmolecules used by researchers and by industry. Today, plasmid DNA isbecoming increasingly important as the next generation of biotechnologyproducts (e.g. gene medicines and DNA vaccines) make their way intoclinical trials, and eventually into the pharmaceutical marketplace.Plasmid DNA vaccines may find application as preventive vaccines forviral, bacterial, or parasitic diseases; immunizing agents for thepreparation of hyper immune globulin products; therapeutic vaccines forinfectious diseases; or as cancer vaccines. Plasmids are also utilizedin gene therapy or gene replacement applications, wherein the desiredgene product is expressed from the plasmid after administration to thepatient.

Therapeutic plasmids often contain a pMB1, ColE1 or pBR322 derivedreplication origin. Common high copy number derivatives have mutationsaffecting copy number regulation, such as ROP (Repressor of primer gene)deletion, with a second site mutation that increases copy number (e.g.pMB1 pUC G to A point mutation, or ColE1 pMM1). Higher temperature (42°C.) can be employed to induce selective plasmid amplification with pUCand pMM1 replication origins.

U.S. Pat. No. 7,943,377 (Carnes, A E and Williams, J A, 2011) disclosemethods for fed-batch fermentation, in which plasmid-containing E. colicells were grown at a reduced temperature during part of the fed-batchphase, during which growth rate was restricted, followed by atemperature up-shift and continued growth at elevated temperature inorder to accumulate plasmid; the temperature shift at restricted growthrate improved plasmid yield and purity. Other fermentation processes forplasmid production are described in Carnes A. E. 2005 BioProcess Intl3:36-44, which is incorporated herein by reference in its entirety.

The art teaches that one of the limitations of application of plasmidtherapies and plasmid vaccines is regulatory agency (e.g. Food and DrugAdministration, European Medicines Agency) safety concerns regarding 1)plasmid transfer and replication in endogenous bacterial flora, or 2)plasmid encoded selection marker expression in human cells, orendogenous bacterial flora. Additionally, regulatory agency guidance'srecommend removal of all non essential sequences in a vector. Plasmidscontaining a pMB1, ColE1 or pBR322 derived replication origin canreplicate promiscuously in E. coli hosts. This presents a safety concernthat a plasmid therapeutic gene or antigen will be transferred andreplicated to a patient's endogenous flora. Ideally, a therapeutic orvaccine plasmid would be replication incompetent in endogenous E. colistrains. This requires replacement of the pMB1, ColE1 or pBR322 derivedreplication origin with a conditional replication origin that requires aspecialized cell line for propagation. As well, regulatory agencies suchas the EMEA and FDA are concerned with utilization of antibioticresistance or alternative protein markers in gene therapy and genevaccine vectors, due to concerns that the gene (antibiotic resistancemarker or protein marker) may be expressed in a patients cells. Ideally,plasmid therapies and plasmid vaccines would: 1) be replicationincompetent in endogenous E. coli strains, 2) not encode a protein basedselection marker and 3) be minimalized to eliminate all non essentialsequences.

The art further teaches that one of the limitations of application ofplasmid therapies and vaccines is that transgene expression is generallyvery low. Vector modifications that improve antigen expression (e.g.codon optimization of the gene, inclusion of an intron, use of thestrong constitutive CMV or CAG promoters versus weaker or cell linespecific promoter) are highly correlative with improved in vivoexpression and, where applicable, immune responses (reviewed in Manoj S,Babiuk L A, van Drunen Little-van den Hurk S. 2004 Crit Rev Clin Lab Sci41: 1-39). A hybrid CMV promoter (CMV/R), which increased antigenexpression, also improved cellular immune responses to HIV DNA vaccinesin mice and nonhuman primates (Barouch D H, Yang Z Y, Kong W P,Korioth-Schmitz B, Sumida S M, Truitt D M, Kishko M G, Arthur J C, MiuraA, Mascola J R, Letvin N L, Nabel G J. 2005 J Virol. 79: 8828-8834). Aplasmid containing the woodchuck hepatitis virus posttranscriptionalregulatory element (a 600 bp element that increases stability andextranuclear transport of RNA resulting in enhanced levels of mRNA fortranslation) enhanced antigen expression and protective immunity toinfluenza hemagglutinin (HA) in mice (Garg S, Oran A E, Hon H, Jacob J.2004 J Immunol. 173: 550-558). These studies teach that improvement inexpression beyond that of current CMV based vectors may generallyimprove immunogenicity and, in the case of gene therapeutics, efficacy.

Transgene expression duration from plasmid vectors is reduced due topromoter inactivation mediated by the bacterial region (i.e. regionencoding bacterial replication origin and selectable marker which isencoded in the spacer region) of the vector (Chen Z Y, He C Y, Meuse L,Kay M A. 2004. Gene Ther 11:856-864; Suzuki M, Kasai K, Saeki Y. 2006. JVirol 80:3293-3300). This results in short duration transgeneexpression. A strategy to improve transgene expression duration is toremove the bacterial region of the plasmid. For example, minicircle and‘linear Minimalistic immunogenic defined gene expression’ (MIDGE)vectors have been developed which do not contain a bacterial region.Removal of the bacterial region in minicircle vectors improved transgeneexpression duration (Chen et al., Supra, 2004). In minicircle vectors,the eukaryotic region polyadenylation signal is covalently linked to theeukaryotic region promoter. This linkage (spacer region) can tolerate aspacer sequence of at least 500 bp since in vivo expression duration isimproved with plasmid vectors in which the bacterial region is removedor replaced with a spacer sequence (spacer region) up to 500 bp inlength (Lu J, Zhang F, Xu S, Fire A Z, Kay M A. 2012. Mol Ther.20:2111-9).

However, methods to manufacture MIDGE and minicircle vectors areexpensive and not easily scalable. Creating terminal loops on MIDGEvectors in vitro is problematic, requiring in vitro ligation of annealedprimers to restriction digested vector. For minicircle vectors, E. colibased manufacturing systems have been developed in which, after plasmidproduction, the bacterial region and the eukaryotic region are separatedand circularized into a minicircle (eukaryotic region) and a bacterialregion circle via the action of phage recombinases on recognitionsequences in the plasmid. In some methods, a restriction enzyme is thenutilized to digest the bacterial region circle at a unique site toeliminate this difficult to remove contaminant. These productionprocedures are very inefficient. For example, optimal manufacture ofminicircle vectors yields only 5 mg of minicircle per liter culture (KayM A, He C Y, Chen Z Y. 2010. Nat Biotechnol 28:1287-1289).

A solution is needed to develop eukaryotic expression vectors thatcontain short spacer regions preferably less than 500 bp that can beefficiently manufactured. These vectors should not encode a proteinbased selection marker and should be minimalized to eliminate all nonessential sequences.

SUMMARY OF THE INVENTION

The present invention relates to a family of minimalized eukaryoticexpression plasmids with short spacer regions that preferably arereplication incompetent in endogenous flora and surprisingly havedramatically improved in vivo expression and high yield manufacture.These vectors are useful for gene therapy, genetic immunization and orinterferon therapy.

Improved vector methods and compositions that utilize novel spacerregion encoded bacterial propagation and selection sequences aredisclosed.

Improved vector methods and compositions that utilize novel spacerregion encoded bacterial propagation sequences with selection sequencesencoded within the eukaryotic expression cassette are disclosed.

Improved vector methods and compositions that utilize novel spacerregion encoded bacterial selection sequences with propagation sequencesencoded within the eukaryotic expression cassette are disclosed.

Improved vector methods and compositions that utilize novel intronicbacterial regions in which bacterial propagation and selection sequencesare encoded within an intron within the eukaryotic expression cassetteare disclosed.

Improved vector methods and compositions that utilize novel bacterialselection sequences encoded within an intron while propagation sequencesare encoded within the spacer region or within the 3′ UTR of theeukaryotic expression cassette are disclosed.

Improved vector methods and compositions that utilize novel bacterialreplication sequences encoded within an intron while selection sequencesare encoded within the spacer region or within the 3′ UTR of theeukaryotic expression cassette are disclosed.

Improved vector methods and compositions that utilize novel 3′UTRbacterial regions in which bacterial propagation and selection sequencesare encoded within a 3′ UTR within the eukaryotic expression cassetteare disclosed.

Improved vector methods and compositions that utilize novel bacterialselection sequences encoded within an 3′UTR while propagation sequencesare encoded within the spacer region or within an intron of theeukaryotic expression cassette are disclosed.

Improved vector methods and compositions that utilize novel intronicbacterial replication sequences encoded within an 3′UTR while selectionsequences are encoded within the spacer region or within an intron ofthe eukaryotic expression cassette are disclosed.

Improved vector methods and compositions wherein a bacterial replicationorigin and a RNA selectable marker are not both positioned within asingle intron, spacer region or 3′UTR are disclosed. In these improvedvectors the replication origin and RNA selection marker are positionedseparately (i.e. without the other) in either an intron, a 3′ UTR or aspacer region. For example, in one embodiment the replication origin ispositioned in the spacer region and the RNA selection marker ispositioned in a intron. For example, in one embodiment the replicationorigin is positioned in the spacer region and the RNA selection markeris positioned in a 3′ UTR. For example, in one embodiment thereplication origin is positioned in a intron and the RNA selectionmarker is positioned in a second intron. For example, in one embodimentthe replication origin is positioned in a intron and the RNA selectionmarker is positioned in a 3′ UTR. For example, in one embodiment thereplication origin is positioned in a intron and the RNA selectionmarker is positioned in a spacer region. For example, in one embodimentthe replication origin is positioned in a 3′UTR and the RNA selectionmarker is positioned in a intron. For example, in one embodiment thereplication origin is positioned in a 3′UTR and the RNA selection markeris positioned in a spacer region.

One object of the invention is to provide improved transgene expressionplasmid vectors.

Another object of the invention is to provide eukaryotic expressionvectors containing short spacer regions less than 500 bp that may beefficiently manufactured.

According to one object of the invention, a method of increasingexpression from an expression plasmid vector comprises modifying theplasmid DNA to replace the pMB1, ColE1 or pBR322 derived replicationorigin and selectable marker with an alternative replication originselected from the group consisting of a R6K gamma replication origin, aColE2-P9 replication origin, and a ColE2-P9 related replication originand an RNA selectable marker; transforming the modified plasmid DNA intoa bacterial cell line rendered competent for transformation; andisolating the resultant transformed bacterial cells. The resultantplasmid surprisingly has higher in vivo expression levels than theparent pMB1, ColE1 or pBR322 derived replication origin expressionplasmid vector.

According to one object of the invention, a composition for constructionof a eukaryotic expression vector comprises an R6K origin with at least90% sequence identity to the sequence set forth as SEQ ID NO: 11 or SEQID NO: 12, and a RNA selectable marker, wherein said R6K origin isoperably linked to said RNA selectable marker and a eukaryotic region.According to still another object of the invention, the RNA selectablemarker is selected from the group consisting of: an RNA-OUT selectablemarker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95%sequence identity to SEQ ID NO: 21; an RNAI selectable marker thatencodes an RNAII regulating RNAI RNA with at least 95% sequence identityto SEQ ID NO: 33; an IncB RNAI selectable marker encoding an RNAIIregulating RNAI RNA with at least 95% sequence identity to SEQ ID NO:35; an synthetic RNA selectable marker encoding an RNA selectable markercomplement regulating RNA with at least 95% sequence identity to SEQ IDNO: 38. According to still another object of the invention, a RNA-OUTselectable marker selected from the group consisting of: SEQ ID NO: 20and SEQ ID NO: 22 is incorporated into the vector adjacent to the R6Korigin. According to still another object of the invention, the RNA-OUTselectable marker—R6K origin is selected from the group consisting of:SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. According to stillanother object of the invention, the synthetic RNA selectable marker—R6Korigin is selected from the group consisting of: SEQ ID NO: 70, SEQ IDNO: 72. According to still another object of the invention, the RNAIselectable marker—R6K origin is selected from the group consisting of:SEQ ID NO: 71, SEQ ID NO: 73. According to another object of theinvention, said R6K origin-RNA selectable marker improves said vectorexpression in vivo compared to a corresponding vector containing a pMB1,ColE1 or pBR322 derived replication origin. According to still anotherobject of the invention, said vector has at least 95% sequence identityto a sequence selected from the group consisting of: SEQ ID NO: 62, SEQID NO: 64, SEQ ID NO:65, SEQ ID NO:66.

According to one object of the invention, a composition for constructionof a eukaryotic expression vector comprises a ColE2-P9 origin with atleast 90% sequence identity to the sequence set forth as SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, and a a RNA selectablemarker, wherein said ColE2-P9 origin—a RNA selectable marker is operablylinked to a eukaryotic region. According to another object of theinvention, said ColE2-P9 origin-RNA selectable marker improves saidvector expression in vivo compared to a corresponding vector containinga pMB1, ColE1 or pBR322 derived replication origin. According to stillanother object of the invention, a primosomal assembly site (ssiA) isoptionally incorporated into the vector adjacent to the ColE2-P9 origin.According to still another object of the invention, a RNA selectablemarker is incorporated into the vector adjacent to the ColE2-P9replication origin. According to still another object of the invention,the RNA selectable marker is selected from the group consisting of: anRNA-OUT selectable marker that encodes an RNA-IN regulating RNA-OUT RNAwith at least 95% sequence identity to SEQ ID NO: 21; an RNAI selectablemarker that encodes an RNAII regulating RNAI RNA with at least 95%sequence identity to SEQ ID NO: 33; an IncB RNAI selectable markerencoding an RNAII regulating RNAI RNA with at least 95% sequenceidentity to SEQ ID NO: 35; an synthetic RNA selectable marker encodingan RNA selectable marker complement regulating RNA with at least 95%sequence identity to SEQ ID NO: 38. According to still another object ofthe invention, a RNA-OUT selectable marker selected from the groupconsisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into thevector adjacent to the ColE2-P9 origin. According to still anotherobject of the invention, the RNA-OUT selectable marker—ColE2-P9 isselected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 24 andSEQ ID NO: 25. According to still another object of the invention, thesynthetic RNA selectable marker—ColE2-P9 origin is selected from thegroup consisting of: SEQ ID NO: 74, SEQ ID NO: 76. According to stillanother object of the invention, the RNAI selectable marker—ColE2-P9origin is selected from the group consisting of: SEQ ID NO: 75, SEQ IDNO: 77. According to still another object of the invention, said vectorhas at least 95% sequence identity to SEQ ID NO: 63, SEQ ID NO: 67, SEQID NO:68, SEQ ID NO: 69.

According to one object of the invention, a method of improvingexpression from an expression plasmid vector comprises modifying theplasmid DNA to replace the spacer region encoded pMB1, ColE1, pBR322,R6K, ColE2-P9 or ColE2-P9 related derived replication origin with analternative intronic encoded replication origin selected from the groupconsisting of an R6K gamma replication origin, a ColE2-P9 replicationorigin, a ColE2-P9 related replication origin, a pUC replication origin,a P_(min) pUC replication origin; transforming the modified plasmid DNAinto a bacterial cell line rendered competent for transformation; andisolating the resultant transformed bacterial cells.

According to one object of the invention, a composition for constructionof a short spacer region eukaryotic expression vector with high yieldmanufacture comprises an R6K origin with at least 90% sequence identityto a sequence selected from the group consisting of: SEQ ID NO: 11 andSEQ ID NO: 12, and a plasmid DNA encoded eukaryotic region, wherein saidR6K origin is operably linked to an intron within said plasmid DNAeukaryotic region. According to still another object of the invention, aRNA selectable marker is incorporated into the vector adjacent to theR6K replication origin. According to still another object of theinvention, a RNA selectable marker is incorporated into the vectorwithin a second intron or within the spacer region or within the 3′ UTR.According to still another object of the invention, the RNA selectablemarker is selected from the group consisting of: an RNA-OUT selectablemarker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95%sequence identity to SEQ ID NO: 21; an RNAI selectable marker thatencodes an RNAII regulating RNAI RNA with at least 95% sequence identityto SEQ ID NO: 33; an IncB RNAI selectable marker encoding an RNAIIregulating RNAI RNA with at least 95% sequence identity to SEQ ID NO:35; an synthetic RNA selectable marker encoding an RNA selectable markercomplement regulating RNA with at least 95% sequence identity to SEQ IDNO: 38. According to still another object of the invention, a RNA-OUTselectable marker selected from the group consisting of: SEQ ID NO: 20and SEQ ID NO: 22 is incorporated into the vector adjacent to the R6Korigin. According to still another object of the invention, a RNA-OUTselectable marker selected from the group consisting of: SEQ ID NO: 20and SEQ ID NO: 22 is incorporated into the vector within a secondintron. According to still another object of the invention, a RNA-OUTselectable marker selected from the group consisting of: SEQ ID NO: 20and SEQ ID NO: 22 is incorporated into the vector within the spacerregion. According to still another object of the invention, a RNA-OUTselectable marker selected from the group consisting of: SEQ ID NO: 20and SEQ ID NO: 22 is incorporated into the vector within a 3′ UTR.According to still another object of the invention, the RNA-OUTselectable marker—R6K origin operably linked to the intron is selectedfrom the group consisting of: SEQ ID NO: 26, SEQ ID NO: 27 and SEQ IDNO: 28. According to still another object of the invention, thesynthetic RNA selectable marker—R6K origin operably linked to the intronis selected from the group consisting of: SEQ ID NO: 70, SEQ ID NO: 72.According to still another object of the invention, the RNAI selectablemarker—R6K origin operably linked to the intron is selected from thegroup consisting of: SEQ ID NO: 71, SEQ ID NO: 73. According to anotherobject of the invention, said intronic R6K origin improves said vectorexpression compared to a corresponding vector containing a pMB1, ColE1or pBR322 derived replication origin encoded in the spacer region.According to still another object of the invention, said eukaryoticregion has at least 95% sequence identity to a sequence selected fromthe group consisting of: SEQ ID NO: 30, SEQ ID NO: 31.

According to one object of the invention, a composition for constructionof a short spacer region eukaryotic expression vector with high yieldmanufacture comprises a ColE2-P9 origin with at least 90% sequenceidentity to a sequence selected from the group consisting of: SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, and a plasmid DNAencoded eukaryotic region, wherein said ColE2-P9 origin is operablylinked to an intron within said plasmid DNA encoded eukaryotic region.According to still another object of the invention, a primosomalassembly site (ssiA) is optionally incorporated into the vector adjacentto the ColE2-P9 origin. According to still another object of theinvention, a RNA selectable marker is incorporated into the vectoradjacent to the ColE2-P9 replication origin. According to still anotherobject of the invention, a RNA selectable marker is incorporated intothe vector within a second intron or within the spacer region or withinthe 3′ UTR. According to still another object of the invention, the RNAselectable marker is selected from the group consisting of: an RNA-OUTselectable marker that encodes an RNA-IN regulating RNA-OUT RNA with atleast 95% sequence identity to SEQ ID NO: 21; an RNAI selectable markerthat encodes an RNAII regulating RNAI RNA with at least 95% sequenceidentity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 35; an synthetic RNA selectable marker encoding an RNA selectablemarker complement regulating RNA with at least 95% sequence identity toSEQ ID NO: 38. According to still another object of the invention, aRNA-OUT selectable marker selected from the group consisting of: SEQ IDNO: 20 and SEQ ID NO: 22 is incorporated into the vector adjacent to theColE2-P9 origin. According to still another object of the invention, aRNA-OUT selectable marker selected from the group consisting of: SEQ IDNO: 20 and SEQ ID NO: 22 is incorporated into the vector within a secondintron. According to still another object of the invention, a RNA-OUTselectable marker selected from the group consisting of: SEQ ID NO: 20and SEQ ID NO: 22 is incorporated into the vector within the spacerregion. According to still another object of the invention, a RNA-OUTselectable marker selected from the group consisting of: SEQ ID NO: 20and SEQ ID NO: 22 is incorporated into the vector within a 3′ UTR.According to still another object of the invention, the RNA-OUTselectable marker—ColE2-P9 origin operably linked to the intron isselected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 24 andSEQ ID NO: 25. According to still another object of the invention, thesynthetic RNA selectable marker—ColE2-P9 origin operably linked to theintron is selected from the group consisting of: SEQ ID NO: 74, SEQ IDNO: 76. According to still another object of the invention, the RNAIselectable marker—ColE2-P9 origin operably linked to the intron isselected from the group consisting of: SEQ ID NO: 75, SEQ ID NO: 77.According to another object of the invention, said intronic ColE2-P9origin improves said vector expression compared to a correspondingvector containing a pMB1, ColE1 or pBR322 derived replication originencoded in the spacer region. According to still another object of theinvention, said eukaryotic region has at least 95% sequence identity toa sequence selected from the group consisting of: SEQ ID NO: 30, SEQ IDNO: 31.

According to one object of the invention, a composition for constructionof a short spacer region eukaryotic expression vector with high yieldmanufacture comprises a pUC origin, and a plasmid DNA encoded eukaryoticregion, wherein said pUC origin is operably linked to an intron withinsaid plasmid DNA eukaryotic region. According to still another object ofthe invention, a RNA selectable marker is incorporated into the vectoradjacent to the pUC replication origin. According to still anotherobject of the invention, a RNA selectable marker is incorporated intothe vector within a second intron or within the spacer region or withinthe 3′ UTR. According to still another object of the invention, the RNAselectable marker is selected from the group consisting of: an RNA-OUTselectable marker that encodes an RNA-IN regulating RNA-OUT RNA with atleast 95% sequence identity to SEQ ID NO: 21; an RNAI selectable markerthat encodes an RNAII regulating RNAI RNA with at least 95% sequenceidentity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 35; an synthetic RNA selectable marker encoding an RNA selectablemarker complement regulating RNA with at least 95% sequence identity toSEQ ID NO: 38. According to still another object of the invention, aRNA-OUT selectable marker selected from the group consisting of: SEQ IDNO: 20 and SEQ ID NO: 22 is incorporated into the vector adjacent to thepUC origin. According to still another object of the invention, aRNA-OUT selectable marker selected from the group consisting of: SEQ IDNO: 20 and SEQ ID NO: 22 is incorporated into the vector within a secondintron. According to still another object of the invention, a RNA-OUTselectable marker selected from the group consisting of: SEQ ID NO: 20and SEQ ID NO: 22 is incorporated into the vector within the spacerregion. According to still another object of the invention, a RNA-OUTselectable marker selected from the group consisting of: SEQ ID NO: 20and SEQ ID NO: 22 is incorporated into the vector within a 3′ UTR.According to still another object of the invention, the RNA-OUTselectable marker—pUC origin operably linked to the intron is SEQ ID NO:29. According to another object of the invention, said intronic pUCorigin improves said vector expression compared to a correspondingvector containing a pMB1, ColE1 or pBR322 derived replication originencoded in the spacer region. According to still another object of theinvention, said eukaryotic region has at least 95% sequence identity toa sequence selected from the group consisting of: SEQ ID NO: 30, SEQ IDNO: 31.

According to one object of the invention, a composition for constructionof a short spacer region eukaryotic expression vector with high yieldmanufacture comprises a P_(min) pUC origin with at least 90% sequenceidentity to SEQ ID NO: 45, and a plasmid DNA encoded eukaryotic region,wherein said P_(min) pUC origin is operably linked to an intron withinsaid plasmid DNA eukaryotic region. According to still another object ofthe invention, a RNA selectable marker is incorporated into the vectoradjacent to the P_(min) pUC replication origin. According to stillanother object of the invention, a RNA selectable marker is incorporatedinto the vector within a second intron or within the spacer region orwithin the 3′ UTR. According to still another object of the invention,the RNA selectable marker is selected from the group consisting of: anRNA-OUT selectable marker that encodes an RNA-IN regulating RNA-OUT RNAwith at least 95% sequence identity to SEQ ID NO: 21; an RNAI selectablemarker that encodes an RNAII regulating RNAI RNA with at least 95%sequence identity to SEQ ID NO: 33; an IncB RNAI selectable markerencoding an RNAII regulating RNAI RNA with at least 95% sequenceidentity to SEQ ID NO: 35; an synthetic RNA selectable marker encodingan RNA selectable marker complement regulating RNA with at least 95%sequence identity to SEQ ID NO: 38. According to still another object ofthe invention, a RNA-OUT selectable marker selected from the groupconsisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into thevector adjacent to the P_(min) pUC origin. According to still anotherobject of the invention, a RNA-OUT selectable marker selected from thegroup consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporatedinto the vector within a second intron. According to still anotherobject of the invention, a RNA-OUT selectable marker selected from thegroup consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporatedinto the vector within the spacer region. According to still anotherobject of the invention, a RNA-OUT selectable marker selected from thegroup consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporatedinto the vector within a 3′ UTR. According to still another object ofthe invention, the RNA-OUT selectable marker—P_(min) pUC origin operablylinked to the intron is SEQ ID NO: 46. According to another object ofthe invention, said intronic P_(min) pUC origin improves said vectorexpression compared to a corresponding vector containing a pMB1, ColE1or pBR322 derived replication origin encoded in the spacer region.According to still another object of the invention, said eukaryoticregion has at least 95% sequence identity to a sequence selected fromthe group consisting of: SEQ ID NO: 30, SEQ ID NO: 31.

According to one object of the invention, a method of improvingexpression from an expression plasmid vector comprises modifying theplasmid DNA to replace the spacer region encoded pMB1, ColE1, pBR322,R6K, ColE2-P9 or ColE2-P9 related derived replication origin with analternative 3′ UTR encoded replication origin selected from the groupconsisting of an R6K gamma replication origin, a ColE2-P9 replicationorigin, a ColE2-P9 related replication origin transforming the modifiedplasmid DNA into a bacterial cell line rendered competent fortransformation; and isolating the resultant transformed bacterial cells.

According to one object of the invention, a composition for constructionof a short spacer region eukaryotic expression vector with high yieldmanufacture comprises an R6K origin with at least 90% sequence identityto a sequence selected from the group consisting of: SEQ ID NO: 11 andSEQ ID NO: 12, and a plasmid DNA encoded eukaryotic region, wherein saidR6K origin is operably linked to a 3′ UTR within said plasmid DNAeukaryotic region. According to still another object of the invention, aRNA selectable marker is incorporated into the vector adjacent to theR6K replication origin. According to still another object of theinvention, a RNA selectable marker is incorporated into the vectorwithin an intron or within the spacer region. According to still anotherobject of the invention, the RNA selectable marker is selected from thegroup consisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. According to stillanother object of the invention, a RNA-OUT selectable marker selectedfrom the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 isincorporated into the vector adjacent to the R6K origin. According tostill another object of the invention, a RNA-OUT selectable markerselected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22is incorporated into the vector within an intron. According to stillanother object of the invention, a RNA-OUT selectable marker selectedfrom the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 isincorporated into the vector within the spacer region. According tostill another object of the invention, the RNA-OUT selectable marker—R6Korigin operably linked to the 3′ UTR is selected from the groupconsisting of: SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. Accordingto still another object of the invention, the synthetic RNA selectablemarker—R6K origin operably linked to the 3′ UTR is selected from thegroup consisting of: SEQ ID NO: 70, SEQ ID NO: 72. According to stillanother object of the invention, the RNAI selectable marker—R6K originoperably linked to the 3′ UTR is selected from the group consisting of:SEQ ID NO: 71, SEQ ID NO: 73. According to another object of theinvention, said 3′ UTR R6K origin improves said vector expressioncompared to a corresponding vector containing a pMB1, ColE1 or pBR322derived replication origin encoded in the spacer region. According tostill another object of the invention, said eukaryotic region has atleast 95% sequence identity to a sequence selected from the groupconsisting of: SEQ ID NO: 30, SEQ ID NO: 31.

According to one object of the invention, a composition for constructionof a short spacer region eukaryotic expression vector with high yieldmanufacture comprises a ColE2-P9 origin with at least 90% sequenceidentity to a sequence selected from the group consisting of: SEQ ID NO:13, 14, 15, or 16, and a plasmid DNA encoded eukaryotic region, whereinsaid ColE2-P9 origin is operably linked to an 3′ UTR within said plasmidDNA encoded eukaryotic region. According to still another object of theinvention, a primosomal assembly site (ssiA) is optionally incorporatedinto the vector adjacent to the ColE2-P9 origin. According to stillanother object of the invention, a RNA selectable marker is incorporatedinto the vector adjacent to the ColE2-P9 replication origin. Accordingto still another object of the invention, a RNA selectable marker isincorporated into the vector within a intron, or within the spacerregion. According to still another object of the invention, the RNAselectable marker is selected from the group consisting of: an RNA-OUTselectable marker that encodes an RNA-IN regulating RNA-OUT RNA with atleast 95% sequence identity to SEQ ID NO: 21; an RNAI selectable markerthat encodes an RNAII regulating RNAI RNA with at least 95% sequenceidentity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 35; an synthetic RNA selectable marker encoding an RNA selectablemarker complement regulating RNA with at least 95% sequence identity toSEQ ID NO: 38. According to still another object of the invention, aRNA-OUT selectable marker selected from the group consisting of: SEQ IDNO: 20 and SEQ ID NO: 22 is incorporated into the vector adjacent to theColE2-P9 origin. According to still another object of the invention, aRNA-OUT selectable marker selected from the group consisting of: SEQ IDNO: 20 and SEQ ID NO: 22 is incorporated into the vector within anintron. According to still another object of the invention, a RNA-OUTselectable marker selected from the group consisting of: SEQ ID NO: 20and SEQ ID NO: 22 is incorporated into the vector within the spacerregion. According to still another object of the invention, the RNA-OUTselectable marker—ColE2-P9 origin operably linked to the 3′ UTR isselected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 24 andSEQ ID NO: 25. According to still another object of the invention, thesynthetic RNA selectable marker—ColE2-P9 origin operably linked to the3′UTR is selected from the group consisting of: SEQ ID NO: 74, SEQ IDNO: 76. According to still another object of the invention, the RNAIselectable marker—ColE2-P9 origin operably linked to the 3′UTR isselected from the group consisting of: SEQ ID NO: 75, SEQ ID NO: 77.According to another object of the invention, said intronic ColE2-P9origin improves said vector expression compared to a correspondingvector containing a pMB1, ColE1 or pBR322 derived replication originencoded in the spacer region. According to still another object of theinvention, said eukaryotic region has at least 95% sequence identity toa sequence selected from the group consisting of: SEQ ID NO: 30, SEQ IDNO: 31.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative minicircle expression vectorcomprising: a. combining, under conditions so as to create a eukaryoticreplicative minicircle expression vector, i) a eukaryotic regionencoding a gene of interest and comprising an intron, a 3′ UTR and 5′and 3′ ends, with ii) a spacer region linking the 5′ and 3′ ends of theeukaryotic region, said spacer region less than 500 basepairs in length,and with iii) a bacterial replication origin and a RNA selectable markerpositioned separately within said intron, 3′ UTR or the spacer regionlinking the 5′ and 3′ ends of the eukaryotic region sequences, whereinsaid bacterial replication origin and said RNA selectable marker are notboth positioned within a single intron, spacer region or 3′ UTR; and b.expressing said gene of interest in said vector, wherein said gene ofinterest in said vector is expressed at a higher level than a vectorcomprising a spacer region greater than 500 basepairs. In a furtherembodiment said RNA selectable marker is an RNA-IN regulating RNA-OUTfunctional variant with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO:20, and SEQ ID NO:22. Ina further embodiment said RNA selectable marker is selected from thegroup consisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. In a further embodimentsaid bacterial replication origin is an R6K replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12. In a further embodiment saidbacterial replication origin is an ColE2-P9 replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16. In a further embodiment said vector has at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ ID NO:47, SEQ ID NO: 48, SEQ ID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51, SEQ IDNO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:56, SEQID NO:57, SEQ ID NO: 58.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative minicircle expression vectorcomprising: a. providing a vector comprising i) a eukaryotic regionencoding a gene of interest and comprising an intron, a 3′ UTR and 5′and 3′ ends and ii) a first spacer region linking the 5′ and 3′ ends ofthe eukaryotic region sequences that encodes a selectable marker and abacterial replication origin, said spacer region greater than 500basepairs in length and capable of expressing said gene of interest at afirst level; b. replacing said first spacer region with a second spacerregion of less than 500 basepairs in length, to produce a modifiedvector wherein a bacterial replication origin and a RNA selectablemarker are positioned separately within said intron, 3′ UTR or thespacer region linking the 5′ and 3′ ends of the eukaryotic regionsequences and are not both positioned within a single intron, spacerregion or 3′ UTR; and c. expressing said gene of interest in saidmodified vector, wherein said gene of interest in said modified vectoris expressed at a higher level than said vector comprising a spacerregion greater than 500 basepairs. In a further embodiment said RNAselectable marker is an RNA-IN regulating RNA-OUT functional variantwith at least 95% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO:20, and SEQ ID NO:22. In a furtherembodiment said RNA selectable marker is selected from the groupconsisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. In a further embodimentsaid bacterial replication origin in said modified vector is an R6Kreplication origin with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12. In afurther embodiment said bacterial replication origin in said modifiedvector is an ColE2-P9 replication origin with at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16. In a further embodimentsaid modified vector has at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO: 58.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative minicircle expression vectorcomprising: a. combining, under conditions so as to create a eukaryoticreplicative minicircle expression vector, i) a eukaryotic regionencoding a gene of interest and comprising an intron, a 3′ UTR and 5′and 3′ ends, with ii) a spacer region linking the 5′ and 3′ ends of theeukaryotic region sequences and comprising a bacterial replicationorigin and a RNA selectable marker, said spacer region less than 500basepairs in length and said bacterial replication origin and said RNAselectable marker positioned separately within said intron, 3′ UTR orthe spacer region linking the 5′ and 3′ ends of the eukaryotic regionsequences and wherein said bacterial replication origin and said RNAselectable marker are not both positioned within a single intron, spacerregion or 3′ UTR; b. transforming said replicative minicircle expressionvector into cells of an RNA selectable marker regulated bacterial cellline; c. isolating the resultant transformed bacterial cells byselection; and d. propagating the resultant transformed bacterial cellsin culture under conditions such as to manufacture said vector in yieldsof greater than 100 mg vector per liter culture. In a further embodimentsaid RNA selectable marker is an RNA-IN regulating RNA-OUT functionalvariant with at least 95% sequence identity to a sequence selected fromthe group consisting of SEQ ID NO:20, and SEQ ID NO:22. In a furtherembodiment said RNA selectable marker is selected from the groupconsisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. In a further embodimentsaid bacterial replication origin is an R6K replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12. In a further embodiment saidbacterial replication origin is an ColE2-P9 replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16. In a further embodiment said vector has at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ ID NO:47, SEQ ID NO: 48, SEQ ID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51, SEQ IDNO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:56, SEQID NO:57, SEQ ID NO: 58.

In one embodiment, the present invention contemplates a eukaryoticreplicative minicircle expression vector comprising i) a eukaryoticregion sequence comprising an intron, a 3′ UTR, and 5′ and 3′ ends andii) a spacer region of less than 500 basepairs in length linking the 5′and 3′ ends of the eukaryotic region sequences and iii) a bacterialreplication origin and a RNA selectable marker positioned separatelywithin an said intron, 3′ UTR or the spacer region linking the 5′ and 3′ends of the eukaryotic region sequences wherein said bacterialreplication origin and said RNA selectable marker are not bothpositioned within a single intron, spacer region or 3′ UTR. In a furtherembodiment said RNA selectable marker is an RNA-IN regulating RNA-OUTfunctional variant with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO:20, and SEQ ID NO:22. Ina further embodiment said RNA selectable marker is selected from thegroup consisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. In a further embodimentsaid bacterial replication origin is an R6K replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12. In a further embodiment saidbacterial replication origin is an ColE2-P9 replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16. In a further embodiment said vector has at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ ID NO:47, SEQ ID NO: 48, SEQ ID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51, SEQ IDNO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:56, SEQID NO:57, SEQ ID NO: 58.

In one embodiment, the present invention contemplates a method ofcontrolling cell growth, comprising: a. providing an isolatedtransformed bacterial host cell comprising: 1) a chromosomal gene whichinhibits cell growth operably linked to a antisense sequence that iscomplementary to a portion of an RNA selectable marker; and 2) aeukaryotic replicative minicircle expression vector comprising i)eukaryotic region sequences comprising an intron, a 3′ UTR, and 5′ and3′ ends; and ii) a spacer region of less than 500 basepairs in lengthlinking the 5′ and 3′ ends of the eukaryotic region sequences and iii) abacterial replication origin and a RNA selectable marker positionedseparately within said intron, 3′ UTR or the spacer region linking the5′ and 3′ ends of the eukaryotic region sequences, wherein saidbacterial replication origin and said RNA selectable marker are not bothpositioned within a single intron, spacer region or 3′ UTR; and b.culturing said bacterial host cell under conditions such that said RNAselectable marker binds to said antisense sequence, wherein binding ofsaid RNA selectable marker to said antisense sequence inhibits theexpression of the chromosomal gene, thereby permitting cell growth. In afurther embodiment said vector has at least 95% sequence identity to asequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO:48, SEQ ID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ IDNO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO: 58. In a further embodiment a method of manufacture comprisingculturing said isolated transformed bacterial host cell in culture mediaunder conditions such that said transformed bacterial host cellmanufactures vector in yields of greater than 100 mg vector per literculture media. In a further embodiment said transformed bacterial hostcell manufactures vector in yields up to 745 mg vector per liter culturemedia. In a further embodiment said RNA selectable marker is an RNA-INregulating RNA-OUT functional variant with at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:20, SEQ ID NO:22. In a further embodiment said RNA selectable markeris selected from the group consisting of: an RNA-OUT selectable markerthat encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequenceidentity to SEQ ID NO: 21; an RNAI selectable marker that encodes anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 33; an IncB RNAI selectable marker encoding an RNAII regulating RNAIRNA with at least 95% sequence identity to SEQ ID NO: 35; an syntheticRNA selectable marker encoding an RNA selectable marker complementregulating RNA with at least 95% sequence identity to SEQ ID NO: 38.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative minicircle expression vectorcomprising: a. combining, under conditions so as to create a eukaryoticreplicative minicircle expression vector, 1) a eukaryotic regionencoding a gene of interest and comprising an intron, a 3′ UTR and 5′and 3′ ends and a bacterial replication origin and a RNA selectablemarker with 2) a spacer region linking the 5′ and 3′ ends of theeukaryotic region of less than 500 basepairs in length and encoding nobacterial sequences, wherein said bacterial replication origin and saidRNA selectable marker positioned separately within said intron or said3′ UTR and wherein said bacterial replication origin and said RNAselectable marker are not both positioned within a single intron, or 3′UTR and neither of said bacterial replication origin and said RNAselectable marker are positioned within said spacer region linking the5′ and 3′ ends of the eukaryotic region sequences; and b. expressingsaid gene of interest in said vector, wherein said gene of interest insaid vector is expressed at a higher level than a vector comprising aspacer region linking the 5′ and 3′ ends of the eukaryotic region ofgreater than 500 basepairs.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative minicircle expression vectorcomprising: a. combining, under conditions so as to create a eukaryoticreplicative minicircle expression vector, 1) a eukaryotic regionencoding a gene of interest and comprising an intron, a 3′ UTR and 5′and 3′ ends and a bacterial replication origin and a RNA selectablemarker with 2) a spacer region linking the 5′ and 3′ ends of theeukaryotic region of less than 500 basepairs in length and encoding nobacterial sequences, wherein said bacterial replication origin and saidRNA selectable marker positioned separately within said intron or said3′ UTR and said bacterial replication origin and said RNA selectablemarker are not both positioned within a single intron, or 3′ UTR andneither of said bacterial replication origin and said RNA selectablemarker are positioned within a spacer region linking the 5′ and 3′ endsof the eukaryotic region sequences; b. transforming said replicativeminicircle expression vector into cells of an RNA selectable markerregulated bacterial cell line; c. isolating the resultant transformedbacterial cells by selection; and d. propagating the resultanttransformed bacterial cells to manufacture said vector in yields ofgreater than 100 mg vector per liter culture.

In one embodiment, the present invention contemplates an isolatedtransformed bacterial host cell comprising: 1) a chromosomal gene whichinhibits cell growth operably linked to a antisense sequence that iscomplementary to a portion of an RNA selectable marker; and 2) aeukaryotic replicative minicircle expression vector comprising i)eukaryotic region sequences comprising an intron, a 3′ UTR, and 5′ and3′ ends; and ii) a spacer region of less than 500 basepairs in lengthlinking the 5′ and 3′ ends of the eukaryotic region sequences and iii) abacterial replication origin and a RNA selectable marker positionedseparately within said intron, 3′ UTR or the spacer region linking the5′ and 3′ ends of the eukaryotic region sequences, wherein saidbacterial replication origin and said RNA selectable marker are not bothpositioned within a single intron, spacer region or 3′ UTR. In a furtherembodiment said vector has at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO: 58.In a further embodiment a method of manufacture comprising culturing theisolated transformed bacterial host cell in culture media underconditions such that said transformed bacterial host cell manufacturesvector in yields of greater than 100 mg vector per liter culture media.In a further embodiment said transformed bacterial host cellmanufactures vector in yields up to 745 mg vector per liter culturemedia. In a further embodiment said RNA selectable marker is an RNA-INregulating RNA-OUT functional variant with at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:20, SEQ ID NO:22. In a further embodiment said RNA selectable markeris selected from the group consisting of: an RNA-OUT selectable markerthat encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequenceidentity to SEQ ID NO: 21; an RNAI selectable marker that encodes anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 33; an IncB RNAI selectable marker encoding an RNAII regulating RNAIRNA with at least 95% sequence identity to SEQ ID NO: 35; an syntheticRNA selectable marker encoding an RNA selectable marker complementregulating RNA with at least 95% sequence identity to SEQ ID NO: 38.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. combining, under conditions so as to create aeukaryotic replicative pUC-free minicircle expression vector, i) aeukaryotic region encoding a gene of interest and comprising an intronand 5′ and 3′ ends, with ii) a spacer region linking the 5′ and 3′ endsof the eukaryotic region, said spacer region less than 500 basepairs inlength, and with iii) a bacterial replication origin that is not the pUCorigin and a RNA selectable marker positioned within said intron; and b.expressing said gene of interest in said vector, wherein said gene ofinterest in said pUC-free vector is expressed at a higher level than avector comprising a pUC origin encoding spacer region greater than 500basepairs. In a further embodiment said RNA selectable marker is anRNA-IN regulating RNA-OUT functional variant with at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:20, and SEQ ID NO:22. In a further embodiment said RNA selectablemarker is selected from the group consisting of: an RNA-OUT selectablemarker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95%sequence identity to SEQ ID NO: 21; an RNAI selectable marker thatencodes an RNAII regulating RNAI RNA with at least 95% sequence identityto SEQ ID NO: 33; an IncB RNAI selectable marker encoding an RNAIIregulating RNAI RNA with at least 95% sequence identity to SEQ ID NO:35; an synthetic RNA selectable marker encoding an RNA selectable markercomplement regulating RNA with at least 95% sequence identity to SEQ IDNO: 38. In a further embodiment said bacterial replication origin is anR6K replication origin with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12. In afurther embodiment said bacterial replication origin is an ColE2-P9replication origin with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16. In a further embodiment said eukaryotic regionhas at least 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 30, SEQ ID NO: 31.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. providing a vector comprising i) a eukaryoticregion encoding a gene of interest and comprising an intron and 5′ and3′ ends and ii) a first spacer region linking the 5′ and 3′ ends of theeukaryotic region sequences that encodes a selectable marker and abacterial replication origin, said spacer region greater than 500basepairs in length and capable of expressing said gene of interest at afirst level; b. replacing said first spacer region with a second spacerregion of less than 500 basepairs in length that does not encode aselectable marker or a bacterial replication origin, c. cloning intosaid intron a bacterial replication origin that is not the pUC originand a RNA selectable marker to produce a modified pUC-free minicircleexpression vector; and d. expressing said gene of interest in saidmodified vector, wherein said gene of interest in said modified vectoris expressed at a higher level than said vector comprising a spacerregion greater than 500 basepairs. In a further embodiment said RNAselectable marker is an RNA-IN regulating RNA-OUT functional variantwith at least 95% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO:20, and SEQ ID NO:22. In a furtherembodiment said RNA selectable marker is selected from the groupconsisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. In a further embodimentsaid bacterial replication origin in said modified vector is an R6Kreplication origin with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12. In afurther embodiment said bacterial replication origin in said modifiedvector is an ColE2-P9 replication origin with at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16. In a further embodimentsaid eukaryotic region has at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. combining, under conditions so as to create aeukaryotic replicative pUC-free minicircle expression vector, i) aeukaryotic region encoding a gene of interest and comprising an intronand 5′ and 3′ ends, with ii) a spacer region linking the 5′ and 3′ endsof the eukaryotic region sequences and a bacterial replication originthat is not the pUC origin and a RNA selectable marker, said spacerregion less than 500 basepairs in length and said bacterial replicationorigin and said RNA selectable marker positioned within said intron; b.transforming said replicative minicircle expression vector into cells ofan RNA selectable marker regulated bacterial cell line; c. isolating theresultant transformed bacterial cells by selection; and d. propagatingthe resultant transformed bacterial cells in culture under conditionssuch as to manufacture said vector in yields of greater than 100 mgvector per liter culture. In a further embodiment said RNA selectablemarker is an RNA-IN regulating RNA-OUT functional variant with at least95% sequence identity to a sequence selected from the group consistingof SEQ ID NO:20, and SEQ ID NO:22. In a further embodiment said RNAselectable marker is selected from the group consisting of: an RNA-OUTselectable marker that encodes an RNA-IN regulating RNA-OUT RNA with atleast 95% sequence identity to SEQ ID NO: 21; an RNAI selectable markerthat encodes an RNAII regulating RNAI RNA with at least 95% sequenceidentity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 35; an synthetic RNA selectable marker encoding an RNA selectablemarker complement regulating RNA with at least 95% sequence identity toSEQ ID NO: 38. In a further embodiment said bacterial replication originis an R6K replication origin with at least 95% sequence identity to asequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:12. In a further embodiment said bacterial replication origin is anColE2-P9 replication origin with at least 95% sequence identity to asequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16. In a further embodiment saideukaryotic region has at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31.

In one embodiment, the present invention contemplates a eukaryoticreplicative pUC-free minicircle expression vector comprising i) aeukaryotic region sequence comprising an intron and 5′ and 3′ ends andii) a spacer region of less than 500 basepairs in length linking the 5′and 3′ ends of the eukaryotic region sequences and iii) a bacterialreplication origin that is not the pUC origin and a RNA selectablemarker positioned within said intron. In a further embodiment said RNAselectable marker is an RNA-IN regulating RNA-OUT functional variantwith at least 95% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO:20, and SEQ ID NO:22. In a furtherembodiment said RNA selectable marker is selected from the groupconsisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. In a further embodimentsaid bacterial replication origin is an R6K replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12. In a further embodiment saidbacterial replication origin is an ColE2-P9 replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16. In a further embodiment said eukaryotic region has at least 95%sequence identity to a sequence selected from the group consisting ofSEQ ID NO: 30, SEQ ID NO: 31.

In one embodiment, the present invention contemplates a method ofcontrolling cell growth, comprising: a. providing an isolatedtransformed bacterial host cell comprising: 1) a chromosomal gene whichinhibits cell growth operably linked to a antisense sequence that iscomplementary to a portion of an RNA selectable marker; and 2) aeukaryotic replicative minicircle expression vector comprising i)eukaryotic region sequences comprising an intron and 5′ and 3′ ends; andii) a spacer region of less than 500 basepairs in length linking the 5′and 3′ ends of the eukaryotic region sequences and iii) a bacterialreplication origin that is not the pUC origin and a RNA selectablemarker positioned within said intron; and b. culturing said bacterialhost cell under conditions such that said RNA selectable marker binds tosaid antisense sequence, wherein binding of said RNA selectable markerto said antisense sequence inhibits the expression of the chromosomalgene, thereby permitting cell growth. In a further embodiment saideukaryotic region has at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31. In afurther embodiment a method of manufacture comprising culturing theisolated transformed bacterial host cell in culture media underconditions such that said transformed bacterial host cell manufacturesvector in yields of greater than 100 mg vector per liter culture media.In a further embodiment said transformed bacterial host cellmanufactures vector in yields up to 745 mg vector per liter culturemedia. In a further embodiment said RNA selectable marker is an RNA-INregulating RNA-OUT functional variant with at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:20, SEQ ID NO:22. In a further embodiment said RNA selectable markeris selected from the group consisting of: an RNA-OUT selectable markerthat encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequenceidentity to SEQ ID NO: 21; an RNAI selectable marker that encodes anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 33; an IncB RNAI selectable marker encoding an RNAII regulating RNAIRNA with at least 95% sequence identity to SEQ ID NO: 35; an syntheticRNA selectable marker encoding an RNA selectable marker complementregulating RNA with at least 95% sequence identity to SEQ ID NO: 38.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. combining, under conditions so as to create aeukaryotic replicative pUC-free minicircle expression vector, 1) aeukaryotic region encoding a gene of interest and comprising an intronand 5′ and 3′ ends and a bacterial replication origin that is not thepUC origin and a RNA selectable marker with 2) a spacer region linkingthe 5′ and 3′ ends of the eukaryotic region of less than 500 basepairsin length and encoding no bacterial sequences and said bacterialreplication origin and said RNA selectable marker are positioned withinsaid intron; and b. expressing said gene of interest in said pUC-freevector, wherein said gene of interest in said vector is expressed at ahigher level than a vector comprising a spacer region linking the 5′ and3′ ends of the eukaryotic region of greater than 500 basepairs.

In one embodiment the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. combining, under conditions so as to create aeukaryotic replicative pUC-free minicircle expression vector, 1) aeukaryotic region encoding a gene of interest and comprising an intronand 5′ and 3′ ends and a bacterial replication origin that is not thepUC origin and a RNA selectable marker with 2) a spacer region linkingthe 5′ and 3′ ends of the eukaryotic region of less than 500 basepairsin length and encoding no bacterial sequences wherein said bacterialreplication origin and said RNA selectable marker are positioned withinsaid intron; b. transforming said replicative pUC-free minicircleexpression vector into cells of an RNA selectable marker regulatedbacterial cell line; c. isolating the resultant transformed bacterialcells by selection; and d. propagating the resultant transformedbacterial cells to manufacture said vector in yields of greater than 100mg vector per liter culture.

In one embodiment, the present invention contemplates a isolatedtransformed bacterial host cell comprising: 1) a chromosomal gene whichinhibits cell growth operably linked to a antisense sequence that iscomplementary to a portion of an RNA selectable marker; and 2) aeukaryotic replicative minicircle expression vector comprising i)eukaryotic region sequences comprising an intron and 5′ and 3′ ends; andii) a spacer region of less than 500 basepairs in length linking the 5′and 3′ ends of the eukaryotic region sequences and iii) a bacterialreplication origin that is not the pUC origin and a RNA selectablemarker positioned within said intron. In a further embodiment theisolated transformed bacterial host cell said eukaryotic region has atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 30, SEQ ID NO: 31. In a further embodiment amethod of manufacture comprising culturing the isolated transformedbacterial host cell in culture media under conditions such that saidtransformed bacterial host cell manufactures vector in yields of greaterthan 100 mg vector per liter culture media. In a further embodiment saidtransformed bacterial host cell manufactures vector in yields up to 745mg vector per liter culture media. In a further embodiment said RNAselectable marker is an RNA-IN regulating RNA-OUT functional variantwith at least 95% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO:20, SEQ ID NO:22. In a further embodimentsaid RNA selectable marker is selected from the group consisting of: anRNA-OUT selectable marker that encodes an RNA-IN regulating RNA-OUT RNAwith at least 95% sequence identity to SEQ ID NO: 21; an RNAI selectablemarker that encodes an RNAII regulating RNAI RNA with at least 95%sequence identity to SEQ ID NO: 33; an IncB RNAI selectable markerencoding an RNAII regulating RNAI RNA with at least 95% sequenceidentity to SEQ ID NO: 35; an synthetic RNA selectable marker encodingan RNA selectable marker complement regulating RNA with at least 95%sequence identity to SEQ ID NO: 38.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. combining, under conditions so as to create aeukaryotic replicative pUC-free minicircle expression vector, i) aeukaryotic region encoding a gene of interest and comprising an 3′ UTRand 5′ and 3′ ends, with ii) a spacer region linking the 5′ and 3′ endsof the eukaryotic region, said spacer region less than 500 basepairs inlength, and with iii) a bacterial replication origin that is not the pUCorigin and a RNA selectable marker positioned within said 3′ UTR; and b.expressing said gene of interest in said pUC-free vector, wherein saidgene of interest in said vector is expressed at a higher level than avector comprising a pUC origin containing spacer region greater than 500basepairs. said RNA selectable marker is an RNA-IN regulating RNA-OUTfunctional variant with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO:20, and SEQ ID NO:22. Ina further embodiment said RNA selectable marker is selected from thegroup consisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. In a further embodimentsaid bacterial replication origin is an R6K replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12. In a further embodiment saidbacterial replication origin is an ColE2-P9 replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16. In a further embodiment said eukaryotic region has at least 95%sequence identity to a sequence selected from the group consisting ofSEQ ID NO: 30, SEQ ID NO: 31.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. providing a vector comprising i) a eukaryoticregion encoding a gene of interest and comprising an 3′ UTR and 5′ and3′ ends and ii) a first spacer region linking the 5′ and 3′ ends of theeukaryotic region sequences that encodes a selectable marker and abacterial replication origin, said spacer region greater than 500basepairs in length and capable of expressing said gene of interest at afirst level; b. replacing said first spacer region with a second spacerregion of less than 500 basepairs in length that does not encode aselectable marker or a bacterial replication origin, c. cloning intosaid 3′ UTR a bacterial replication origin that is not the pUC originand a RNA selectable marker to produce a modified pUC-free minicircleexpression vector; and d. expressing said gene of interest in saidmodified pUC-free vector, wherein said gene of interest in said modifiedvector is expressed at a higher level than said vector comprising aspacer region greater than 500 basepairs. In a further embodiment saidRNA selectable marker is an RNA-IN regulating RNA-OUT functional variantwith at least 95% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO:20, and SEQ ID NO:22. In a furtherembodiment said RNA selectable marker is selected from the groupconsisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. In a further embodimentsaid bacterial replication origin in said modified vector is an R6Kreplication origin with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12. In afurther embodiment said bacterial replication origin in said modifiedvector is an ColE2-P9 replication origin with at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16. In a further embodimentsaid eukaryotic region has at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. combining, under conditions so as to create aeukaryotic pUC-free replicative minicircle expression vector, i) aeukaryotic region encoding a gene of interest and comprising an 3′ UTRand 5′ and 3′ ends, with ii) a spacer region linking the 5′ and 3′ endsof the eukaryotic region sequences and a bacterial replication originthat is not the pUC origin and a RNA selectable marker, said spacerregion less than 500 basepairs in length and said bacterial replicationorigin and said RNA selectable marker positioned within said 3′ UTR; b.transforming said replicative pUC-free minicircle expression vector intocells of an RNA selectable marker regulated bacterial cell line; c.isolating the resultant transformed bacterial cells by selection; and d.propagating the resultant transformed bacterial cells in culture underconditions such as to manufacture said vector in yields of greater than100 mg vector per liter culture. In a further embodiment said RNAselectable marker is an RNA-IN regulating RNA-OUT functional variantwith at least 95% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO:20, and SEQ ID NO:22. In a furtherembodiment said RNA selectable marker is selected from the groupconsisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. In a further embodimentsaid bacterial replication origin is an R6K replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12. In a further embodiment saidbacterial replication origin is an ColE2-P9 replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16. In a further embodiment said eukaryotic region has at least 95%sequence identity to a sequence selected from the group consisting ofSEQ ID NO: 30, SEQ ID NO: 31.

In one embodiment, the present invention contemplates a eukaryoticreplicative pUC-free minicircle expression vector comprising i) aeukaryotic region sequence comprising an 3′ UTR and 5′ and 3′ ends andii) a spacer region of less than 500 basepairs in length linking the 5′and 3′ ends of the eukaryotic region sequences and iii) a bacterialreplication origin that is not the pUC origin and a RNA selectablemarker positioned within said 3′ UTR. In a further embodiment said RNAselectable marker is an RNA-IN regulating RNA-OUT functional variantwith at least 95% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO:20, and SEQ ID NO:22. In a furtherembodiment said RNA selectable marker is selected from the groupconsisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. In a further embodimentsaid bacterial replication origin is an R6K replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12. In a further embodiment saidbacterial replication origin is an ColE2-P9 replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16. In a further embodiment said eukaryotic region has at least 95%sequence identity to a sequence selected from the group consisting ofSEQ ID NO: 30, SEQ ID NO: 31.

In one embodiment, the present invention contemplates a method ofcontrolling cell growth, comprising: a. providing an isolatedtransformed bacterial host cell comprising: 1) a chromosomal gene whichinhibits cell growth operably linked to a antisense sequence that iscomplementary to a portion of an RNA selectable marker; and 2) aeukaryotic replicative minicircle expression vector comprising i)eukaryotic region sequences comprising an 3′ UTR and 5′ and 3′ ends; andii) a spacer region of less than 500 basepairs in length linking the 5′and 3′ ends of the eukaryotic region sequences and iii) a bacterialreplication origin that is not the pUC origin and a RNA selectablemarker positioned within said 3′ UTR; and b. culturing said transformedbacterial host cell under conditions such that said RNA selectablemarker binds to said antisense sequence, wherein binding of said RNAselectable marker to said antisense sequence inhibits the expression ofthe chromosomal gene, thereby permitting cell growth. In a furtherembodiment said eukaryotic region has at least 95% sequence identity toa sequence selected from the group consisting of SEQ ID NO: 30, SEQ IDNO: 31. In a further embodiment a method of manufacture comprisingculturing the isolated transformed bacterial host cell in culture mediaunder conditions such that said transformed bacterial host cellmanufactures vector in yields of greater than 100 mg vector per literculture media. In a further embodiment said transformed bacterial hostcell manufactures vector in yields up to 745 mg vector per liter culturemedia. In a further embodiment said RNA selectable marker is an RNA-INregulating RNA-OUT functional variant with at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:20, SEQ ID NO:22. In a further embodiment said RNA selectable markeris selected from the group consisting of: an RNA-OUT selectable markerthat encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequenceidentity to SEQ ID NO: 21; an RNAI selectable marker that encodes anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 33; an IncB RNAI selectable marker encoding an RNAII regulating RNAIRNA with at least 95% sequence identity to SEQ ID NO: 35; an syntheticRNA selectable marker encoding an RNA selectable marker complementregulating RNA with at least 95% sequence identity to SEQ ID NO: 38.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. combining, under conditions so as to create aeukaryotic replicative pUC-free minicircle expression vector, 1) aeukaryotic region encoding a gene of interest and comprising an 3′ UTRand 5′ and 3′ ends and a bacterial replication origin that is not thepUC origin and a RNA selectable marker with 2) a spacer region linkingthe 5′ and 3′ ends of the eukaryotic region of less than 500 basepairsin length and encoding no bacterial sequences and said bacterialreplication origin and said RNA selectable marker are positioned withinsaid 3′ UTR; and b. expressing said gene of interest in said vector,wherein said gene of interest in said vector is expressed at a higherlevel than a vector comprising a spacer region linking the 5′ and 3′ends of the eukaryotic region of greater than 500 basepairs.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. combining, under conditions so as to create aeukaryotic replicative pUC-free minicircle expression vector, 1) aeukaryotic region encoding a gene of interest and comprising an 3′ UTRand 5′ and 3′ ends and a bacterial replication origin that is not thepUC origin and a RNA selectable marker with 2) a spacer region linkingthe 5′ and 3′ ends of the eukaryotic region of less than 500 basepairsin length and encoding no bacterial sequences wherein said bacterialreplication origin and said RNA selectable marker are positioned withinsaid 3′ UTR; b. transforming said replicative pUC-free minicircleexpression vector into cells of an RNA selectable marker regulatedbacterial cell line; c. isolating the resultant transformed bacterialcells by selection; and d. propagating the resultant transformedbacterial cells to manufacture said vector in yields of greater than 100mg vector per liter culture.

In one embodiment, the present invention contemplates an isolatedtransformed bacterial host cell comprising: 1) a chromosomal gene whichinhibits cell growth operably linked to a antisense sequence that iscomplementary to a portion of an RNA selectable marker; and 2) aeukaryotic replicative minicircle expression vector comprising i)eukaryotic region sequences comprising an 3′ UTR and 5′ and 3′ ends; andii) a spacer region of less than 500 basepairs in length linking the 5′and 3′ ends of the eukaryotic region sequences and iii) a bacterialreplication origin that is not the pUC origin and a RNA selectablemarker positioned within said 3′ UTR. In a further embodiment saideukaryotic region has at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31. In afurther embodiment a method of manufacture comprising culturing theisolated transformed bacterial host cell in culture media underconditions such that said transformed bacterial host cell manufacturesvector in yields of greater than 100 mg vector per liter culture media.In a further embodiment said transformed bacterial host cellmanufactures vector in yields up to 745 mg vector per liter culturemedia. In a further embodiment said RNA selectable marker is an RNA-INregulating RNA-OUT functional variant with at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:20, SEQ ID NO:22. said RNA selectable marker is selected from thegroup consisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. combining, under conditions so as to create aeukaryotic replicative pUC-free minicircle expression vector, i) aeukaryotic region encoding a gene of interest and 5′ and 3′ ends, withii) a spacer region linking the 5′ and 3′ ends of the eukaryotic region,said spacer comprising a bacterial replication origin that is not thepUC origin and a RNA selectable marker, said spacer region less than 500basepairs in length; and b. expressing said gene of interest in saidpUC-free vector, wherein said gene of interest in said pUC-free vectoris expressed at a higher level than a vector comprising a spacer regioncomprising pUC. In a further embodiment said RNA selectable marker is anRNA-IN regulating RNA-OUT functional variant with at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:20, and SEQ ID NO:22. In a further embodiment said RNA selectablemarker is selected from the group consisting of: an RNA-OUT selectablemarker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95%sequence identity to SEQ ID NO: 21; an RNAI selectable marker thatencodes an RNAII regulating RNAI RNA with at least 95% sequence identityto SEQ ID NO: 33; an IncB RNAI selectable marker encoding an RNAIIregulating RNAI RNA with at least 95% sequence identity to SEQ ID NO:35; an synthetic RNA selectable marker encoding an RNA selectable markercomplement regulating RNA with at least 95% sequence identity to SEQ IDNO: 38. In a further embodiment said bacterial replication origin is anR6K replication origin with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12. In afurther embodiment said bacterial replication origin is an ColE2-P9replication origin with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16. In a further embodiment said vector has atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64; SEQ ID NO:65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. providing a vector comprising i) a eukaryoticregion encoding a gene of interest and comprising 5′ and 3′ ends and ii)a first spacer region linking the 5′ and 3′ ends of the eukaryoticregion sequences that encodes a selectable marker and a bacterialreplication origin, said spacer region greater than 500 basepairs inlength and capable of expressing said gene of interest at a first level;b. replacing said first spacer region with a second spacer region ofless than 500 basepairs in length, to produce a pUC-free modified vectorwherein the bacterial replication origin is not the pUC origin and theRNA selectable marker are both positioned within said spacer region; andc. expressing said gene of interest in said pUC-free modified vector,wherein said gene of interest in said pUC-free modified vector isexpressed at a higher level than said vector comprising a spacer regiongreater than 500 basepairs. In a further embodiment said RNA selectablemarker is an RNA-IN regulating RNA-OUT functional variant with at least95% sequence identity to a sequence selected from the group consistingof SEQ ID NO:20, and SEQ ID NO:22. In a further embodiment said RNAselectable marker is selected from the group consisting of: an RNA-OUTselectable marker that encodes an RNA-IN regulating RNA-OUT RNA with atleast 95% sequence identity to SEQ ID NO: 21; an RNAI selectable markerthat encodes an RNAII regulating RNAI RNA with at least 95% sequenceidentity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 35; an synthetic RNA selectable marker encoding an RNA selectablemarker complement regulating RNA with at least 95% sequence identity toSEQ ID NO: 38. In a further embodiment said bacterial replication originin said modified vector is an R6K replication origin with at least 95%sequence identity to a sequence selected from the group consisting ofSEQ ID NO: 11, SEQ ID NO: 12. In a further embodiment said bacterialreplication origin in said modified vector is an ColE2-P9 replicationorigin with at least 95% sequence identity to a sequence selected fromthe group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16. In a further embodiment said modified vector has at least 95%sequence identity to a sequence selected from the group consisting ofSEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64; SEQ ID NO: 65, SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69.

In one embodiment, the present invention contemplates a method ofconstructing a eukaryotic replicative pUC-free minicircle expressionvector comprising: a. combining, under conditions so as to create aeukaryotic replicative pUC-free minicircle expression vector, i) aeukaryotic region encoding a gene of interest and comprising 5′ and 3′ends, with ii) a spacer region linking the 5′ and 3′ ends of theeukaryotic region sequences, said spacer comprising a bacterialreplication origin that is not the pUC origin and a RNA selectablemarker, said spacer region less than 500 basepairs in length; b.transforming said replicative pUC-free minicircle expression vector intocells of an RNA selectable marker regulated bacterial cell line; c.isolating the resultant transformed bacterial cells by selection; and d.propagating the resultant transformed bacterial cells in culture underconditions such as to manufacture said vector in yields of greater than100 mg vector per liter culture. In a further embodiment said RNAselectable marker is an RNA-IN regulating RNA-OUT functional variantwith at least 95% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO:20, and SEQ ID NO:22. In a furtherembodiment said RNA selectable marker is selected from the groupconsisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO: 38. In a further embodimentsaid bacterial replication origin is an R6K replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12. In a further embodiment saidbacterial replication origin is an ColE2-P9 replication origin with atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16. In a further embodiment said vector has at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ ID NO:62, SEQ ID NO: 63, SEQ ID NO: 64; SEQ ID NO: 65, SEQ ID NO: 66, SEQ IDNO: 67, SEQ ID NO: 68, SEQ ID NO: 69.

In one embodiment, the present invention contemplates a eukaryoticreplicative pUC-free minicircle expression vector comprising i) aeukaryotic region sequence comprising 5′ and 3′ ends and ii) a spacerregion of less than 500 basepairs in length linking the 5′ and 3′ endsof the eukaryotic region sequences and comprising a bacterialreplication origin that is not the pUC origin and a RNA selectablemarker. In a further embodiment said RNA selectable marker is an RNA-INregulating RNA-OUT functional variant with at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:20, and SEQ ID NO:22. In a further embodiment said RNA selectablemarker is selected from the group consisting of: an RNA-OUT selectablemarker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95%sequence identity to SEQ ID NO: 21; an RNAI selectable marker thatencodes an RNAII regulating RNAI RNA with at least 95% sequence identityto SEQ ID NO: 33; an IncB RNAI selectable marker encoding an RNAIIregulating RNAI RNA with at least 95% sequence identity to SEQ ID NO:35; an synthetic RNA selectable marker encoding an RNA selectable markercomplement regulating RNA with at least 95% sequence identity to SEQ IDNO: 38. In a further embodiment said bacterial replication origin is anR6K replication origin with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12. In afurther embodiment said bacterial replication origin is an ColE2-P9replication origin with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16. In a further embodiment said vector has atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64; SEQ ID NO:65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69.

In one embodiment, the present invention contemplates a method ofcontrolling cell growth, comprising: a. providing an isolatedtransformed bacterial host cell comprising: 1) a chromosomal gene whichinhibits cell growth operably linked to a antisense sequence that iscomplementary to a portion of an RNA selectable marker; and 2) aeukaryotic replicative minicircle expression vector comprising i)eukaryotic region sequences comprising 5′ and 3′ ends; and ii) a spacerregion of less than 500 basepairs in length linking the 5′ and 3′ endsof the eukaryotic region sequences and comprising a bacterialreplication origin that is not the pUC origin and a RNA selectablemarker; and b. culturing said bacterial host cell under conditions suchthat said RNA selectable marker binds to said antisense sequence,wherein binding of said RNA selectable marker to said antisense sequenceinhibits the expression of the chromosomal gene, thereby permitting cellgrowth. In a further embodiment said vector has at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ ID NO:62, SEQ ID NO: 63, SEQ ID NO: 64; SEQ ID NO: 65, SEQ ID NO: 66, SEQ IDNO: 67, SEQ ID NO: 68, SEQ ID NO: 69. In a further embodiment a methodof manufacture comprising culturing the isolated transformed bacterialhost cell in culture media under conditions such that said transformedbacterial host cell manufactures vector in yields of greater than 100 mgvector per liter culture media. In a further embodiment said transformedbacterial host cell manufactures vector in yields up to 745 mg vectorper liter culture media. In a further embodiment said RNA selectablemarker is an RNA-IN regulating RNA-OUT functional variant with at least95% sequence identity to a sequence selected from the group consistingof SEQ ID NO:20, SEQ ID NO:22. In a further embodiment said RNAselectable marker is selected from the group consisting of: an RNA-OUTselectable marker that encodes an RNA-IN regulating RNA-OUT RNA with atleast 95% sequence identity to SEQ ID NO: 21; an RNAI selectable markerthat encodes an RNAII regulating RNAI RNA with at least 95% sequenceidentity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 35; an synthetic RNA selectable marker encoding an RNA selectablemarker complement regulating RNA with at least 95% sequence identity toSEQ ID NO: 38.

In one embodiment, the present invention contemplates an isolatedtransformed bacterial host cell comprising: 1) a chromosomal gene whichinhibits cell growth operably linked to a antisense sequence that iscomplementary to a portion of an RNA selectable marker; and 2) aeukaryotic replicative minicircle expression vector comprising i)eukaryotic region sequences comprising 5′ and 3′ ends; and ii) a spacerregion of less than 500 basepairs in length linking the 5′ and 3′ endsof the eukaryotic region sequences and comprising a bacterialreplication origin that is not the pUC origin and a RNA selectablemarker. In a further embodiment said vector has at least 95% sequenceidentity to a sequence selected from the group consisting of SEQ ID NO:62, SEQ ID NO: 63, SEQ ID NO: 64; SEQ ID NO: 65, SEQ ID NO: 66, SEQ IDNO: 67, SEQ ID NO: 68, SEQ ID NO: 69. In a further embodiment a methodof manufacture comprising culturing the isolated transformed bacterialhost cell in culture media under conditions such that said transformedbacterial host cell manufactures vector in yields of greater than 100 mgvector per liter culture media. In a further embodiment said transformedbacterial host cell manufactures vector in yields up to 745 mg vectorper liter culture media. In a further embodiment said RNA selectablemarker is an RNA-IN regulating RNA-OUT functional variant with at least95% sequence identity to a sequence selected from the group consistingof SEQ ID NO:20, SEQ ID NO:22. In a further embodiment said RNAselectable marker is selected from the group consisting of: an RNA-OUTselectable marker that encodes an RNA-IN regulating RNA-OUT RNA with atleast 95% sequence identity to SEQ ID NO: 21; an RNAI selectable markerthat encodes an RNAII regulating RNAI RNA with at least 95% sequenceidentity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 35; an synthetic RNA selectable marker encoding an RNA selectablemarker complement regulating RNA with at least 95% sequence identity toSEQ ID NO: 38.

Further objects and advantages of the invention will become apparentfrom a consideration of the drawings and ensuing description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the NTC8485 pUC origin expression vector;

FIG. 2 depicts bioinformatics analysis of an intron containing the gWIZbacterial region (GBR) encoded kanR selection marker-pUC origin;

FIG. 3 depicts bioinformatics analysis of introns containing theNTC9385P2 bacterial region (P2) encoded RNA-OUT selectable marker—pUCorigin in both orientations;

FIG. 4 depicts the NTC9385P2a-O1-EGFP and NTC9385P2a-O2-EGFP intronicpUC origin-RNA-OUT replicative minicircle expression vectors;

FIG. 5 shows plasmid quality from intronic pUC origin-RNA-OUT expressionvectors NTC9385P2a-O1-EGFP, NTC9385P2a-O2-EGFP, NTC9385P2-O1-EGFP andNTC9385P2-O2-EGFP vectors versus a comparator spacer region encoded pUCorigin-RNA-OUT expression vector NTC8385-EGFP;

FIG. 6 depicts the NTC9385R2a-O1-EGFP and NTC9385R2a-O2-EGFP intronicR6K origin-RNA-OUT replicative minicircle expression vectors;

FIG. 7 depicts the NTC9385C2a-O1-EGFP and NTC9385C2a-O2-EGFP intronicColE2 origin-RNA-OUT replicative minicircle expression vectors;

FIG. 8 shows plasmid quality from Table 7 fermentations of intronic R6Korigin-RNA-OUT expression vectors NTC9385R2-O1-EGFP, NTC9385R2-O2-EGFP,NTC9385R2a-O1-EGFP and NTC9385R2a-O2-EGFP vectors, versus a comparatorspacer region encoded R6K origin-RNA-OUT expression vectorNTC9385R-EGFP;

FIG. 9 depicts the NTC9385R2b-O2-EGFP intronic R6K origin-spacer regionRNA-OUT replicative minicircle expression vectors;

FIG. 10 depicts a pMB1 and ColE1 RNA I RNA selectable marker;

FIG. 11 depicts a IncB RNAI based RNA selectable marker;

FIG. 12 depicts a designed synthetic CpG free RNA selectable marker; and

FIG. 13 depicts the NTC9385RbF-EGFP 3′ UTR R6K, intronic RNA-OUTreplicative minicircle expression vector.

Table 1: gWIZ, NTC9385C and NTC9385R Nanoplasmid expression compared toNTC8685

Table 2: Intron encoded RNA-OUT selection/replication origin does notprevent transgene expression;

Table 3: Improved expression with intron encoded RNA-OUTselection/replication origin;

Table 4: Intron functional analysis—Splicing accuracy and exportefficiency;

Table 5: Intron vector expression efficiency;

Table 6: Replicative minicircle vector expression in vitro(lipofectamine) and in vivo (intradermal delivery with electroporation);

Table 7: Intronic RNA-OUT AF selection plasmid fermentation yields;

Table 8: High level expression with vectors with pMB1 RNAI encoded inthe spacer region or intron;

Table 9: Accurate splicing with replicative minicircle vectors with pMB1RNAI and minimal pUC origin encoded in the intron;

Table 10: SR vector expression in vitro and in vivo;

Table 11: Robust expression with P2 (0.85) replicative minicircles;

Table 12:High level expression with R6K replication origin and/orRNA-OUT encoded in the 3′ UTR;

Table 13: High level expression with R6K replication origin encoded inthe 3′ UTR;

Table 14: High level expression with RNAI encoded in the 3′ UTR;

Table 15: Spacer region, intron or 3′ UTR encoded RSMselection/replication origin short spacer region replicative minicirclevector configurations; and

Table 16: Spacer region, intron or 3′ UTR encoded separated RSMselection/replication origin short spacer region replicative minicirclevector configurations.

SEQ ID NO:1: HTLV-IR-Rabbit β globin hybrid intron

SEQ ID NO:2: HTLV-IR CMV hybrid intron

SEQ ID NO:3: CMV intron

SEQ ID NO:4: CpG free intron I 140

SEQ ID NO:5: Human β globin Murine IgG chimeric intron

SEQ ID NO:6: Adenovirus leader-Murine IgG chimeric intron

SEQ ID NO:7: Rabbit β globin intron

SEQ ID NO:8: Truncated CMV intron

SEQ ID NO:9: CAG (Chicken β Actin-rabbit β globin) intron

SEQ ID NO:10: CMV-Rabbit β globin hybrid intron

SEQ ID NO:11: R6K gamma origin

SEQ ID NO:12: CpG free R6K gamma origin

SEQ ID NO:13: ColE2 Origin (+7)

SEQ ID NO:14: ColE2 Origin (Min)

SEQ ID NO:15: ColE2 origin (Core)

SEQ ID NO:16: CpG free ColE2 Origin (+7, CpG free)

SEQ ID NO:17: CpG free ssiA [from plasmid R6K]

SEQ ID NO:18: +7(CpG free) ColE2 origin-CpG free ssiA

SEQ ID NO:19: +7(CpG free) ColE2 origin-CpG free ssiA-flanked by SphIand KpnI restriction sites

SEQ ID NO:20: RNA-OUT Selectable Marker

SEQ ID NO:21: RNA-OUT antisense repressor RNA

SEQ ID NO:22: CpG free RNA-OUT RNA selectable marker

SEQ ID NO:23: RNA-OUT-ColE2 origin bacterial region. [NheIsite-ssiA-ColE2 Origin (+7)-RNA-OUT-KpnI site]

SEQ ID NO:24: NTC9385C2 and NTC9385C2a intronic bacterial region.[filled NheI site-ssiA-ColE2 Origin (+7)-RNA-OUT-chewed KpnI site]Sequence show is O1; O2 is reverse complement

SEQ ID NO:25: CpG free ColE2 RNA-OUT bacterial region. (CpG freessiA-CpG free ColE2 origin-CpG free RNA-OUT RNA selectablemarker)—flanked by SphI and BglII restriction sites

SEQ ID NO:26: RNA-OUT-R6K gamma origin bacterial region. [NheI site-trpAterminator-R6K Origin-RNA-OUT-KpnI site]

SEQ ID NO:27: NTC9385R2 and NTC9385R2a intronic R6K gamma origin-RNA-OUTbacterial region. [filled NheI site-trpA terminator-R6KOrigin-RNA-OUT-chewed KpnI site] Sequence show is O1; O2 is reversecomplement

SEQ ID NO:28: CpG free R6K gamma origin RNA-OUT bacterial region.Flanked by SphI and BglII restriction sites

SEQ ID NO:29: NTC9385P2 and NTC9385P2a intronic pUC origin-RNA-OUTBacterial region. [filled NheI site-trpA terminator-pUCOrigin-RNA-OUT-chewed KpnI site] Sequence show is O1; O2 is reversecomplement

SEQ ID NO:30: NTC9385C2, NTC9385R2, NTC9385P2, NTC9385P2(0.85)Eukaryotic region. Bp 1 is start of CMV enhancer, bp 1196 is end ofpolyadenylation signal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT)transgene cloning sites. Intron encoded HpaI (GTTAAC) bacterial regioncloning site

SEQ ID NO:31: NTC9385C2a, NTC9385R2a, NTC9385P2a and NTC9385P2a(0.85)Eukaryotic region. Bp 1 is start of CMV enhancer encoded boundaryregion, bp 1292 is end of polyadenylation signal. Exon 2 encoded SalI(GTCGAC) and BglII (AGATCT) transgene cloning sites. Intron encoded HpaI(GTTAAC) bacterial region cloning site

SEQ ID NO:32: CpG free HTLV-IR-Rabbit β globin hybrid intron

SEQ ID NO:33: RNAI antisense repressor RNA (pMB 1 plasmid origin RNAIIantisense partner)

SEQ ID NO:34: RNAI selectable Marker

SEQ ID NO:35: IncB RNAI antisense repressor RNA (IncB plasmid originRNAII antisense partner)

SEQ ID NO:36: IncB RNAI selectable Marker, RNAI RNA

SEQ ID NO:37: IncB RNAII-SacB, PstI-MamI restriction fragment

SEQ ID NO:38: RNA selectable marker (RSM) antisense repressor RNA

SEQ ID NO:39: RNA selectable marker (RSM)

SEQ ID NO:40: RSM complement

SEQ ID NO:41: RNA selection-sacB (P5/6 4/6)

SEQ ID NO:42: RNA selection-sacB (P5/6 5/6)

SEQ ID NO:43: pINT-RNAS integration vector (P5/6 4/6)

SEQ ID NO:44: pINT-RNAS integration vector (P5/6 5/6)

SEQ ID NO:45: P_(min) pUC replication origin (minimal)

SEQ ID NO:46: NTC9385P2(0.85) and NTC9385P2a(0.85) intronic pUC (0.85)Bacterial region [[filled NheI site-trpA terminator-P_(min) pUCreplication origin-RNA-OUT-chewed KpnI site] Sequence shown is O1; O2 isreverse complement

SEQ ID NO:47: NTC9385RbF vector backbone. Bp 1 is start of CMV enhancer,last bp is end of polyadenylation signal. Exon 2 encoded SalI (GTCGAC)and BglII (AGATCT) transgene cloning sites are juxtaposed

SEQ ID NO:48: NTC9385RbF-RSM vector backbone. Bp 1 is start of CMVenhancer, last bp is end of polyadenylation signal. Exon 2 encoded SalI(GTCGAC) and BglII (AGATCT) transgene cloning sites are juxtaposed

SEQ ID NO:49: NTC9385RbF-RNAI vector backbone. Bp 1 is start of CMVenhancer, last bp is end of polyadenylation signal. Exon 2 encoded SalI(GTCGAC) and BglII (AGATCT) transgene cloning sites are juxtaposed

SEQ ID NO:50: NTC9385Ra-O1 vector backbone. Bp 1 is start of trpAterminator upstream of R6K origin, last bp is end of polyadenylationsignal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgenecloning sites are juxtaposed

SEQ ID NO:51: NTC9385Ra-O2 vector backbone. Bp 1 is start of trpAterminator upstream of R6K origin, last bp is end of polyadenylationsignal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgenecloning sites are juxtaposed

SEQ ID NO:52: NTC9385Ra-O1-RSM vector backbone. Bp 1 is start of trpAterminator upstream of R6K origin, last bp is end of polyadenylationsignal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgenecloning sites are juxtaposed

SEQ ID NO:53: NTC9385Ra-O2-RSM vector backbone. Bp 1 is start of trpAterminator upstream of R6K origin, last bp is end of polyadenylationsignal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgenecloning sites are juxtaposed

SEQ ID NO:54: NTC9385Ra-O1-RNAI vector backbone. Bp 1 is start of trpAterminator upstream of R6K origin, last bp is end of polyadenylationsignal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgenecloning sites are juxtaposed

SEQ ID NO:55: NTC9385Ra-O2-RNAI vector backbone. Bp 1 is start of trpAterminator upstream of R6K origin, last bp is end of polyadenylationsignal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgenecloning sites are juxtaposed

SEQ ID NO:56: NTC9385RaF vector backbone. Bp 1 is start of trpAterminator upstream of R6K origin, last bp is end of polyadenylationsignal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgenecloning sites are juxtaposed

SEQ ID NO:57: NTC9385RaF-RSM vector backbone. Bp 1 is start of trpAterminator upstream of R6K origin, last bp is end of polyadenylationsignal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgenecloning sites are juxtaposed

SEQ ID NO:58: NTC9385RaF-RNAI vector backbone. Bp 1 is start of trpAterminator upstream of R6K origin, last bp is end of polyadenylationsignal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgenecloning sites are juxtaposed

SEQ ID NO:59: Anti-RNAI (10-108)

SEQ ID NO:60: Anti-RNAI (10-108)-weak RBS-ATG

SEQ ID NO:61: Anti-RNAI (10-108)-strong RBS-ATG

SEQ ID NO:62: NTC9385R vector backbone. Bp 1 is start of trpA terminatorupstream of R6K origin, last bp is end of polyadenylation signal. Exon 2encoded SalI (GTCGAC) and BglII (AGATCT) transgene cloning sites arejuxtaposed

SEQ ID NO:63: NTC9385C vector backbone. Bp 1 is start of ssiA upstreamof ColE2 origin, last bp is end of polyadenylation signal. Exon 2encoded SalI (GTCGAC) and BglII (AGATCT) transgene cloning sites arejuxtaposed

SEQ ID NO:64: NTC9385R-intron vector backbone. Bp 1 is start of trpAterminator upstream of R6K origin, last bp is end of polyadenylationsignal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgenecloning sites are juxtaposed

SEQ ID NO:65: NTC9385R-intron RSM vector backbone. Bp 1 is start of trpAterminator upstream of R6K origin, last bp is end of polyadenylationsignal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgenecloning sites are juxtaposed

SEQ ID NO:66: NTC9385R-intron RNAI vector backbone. Bp 1 is start oftrpA terminator upstream of R6K origin, last bp is end ofpolyadenylation signal. Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT)transgene cloning sites are juxtaposed

SEQ ID NO:67: NTC9385C-intron vector backbone. Bp 1 is start of ssiAupstream of ColE2 origin, last bp is end of polyadenylation signal. Exon2 encoded SalI (GTCGAC) and BglII (AGATCT) transgene cloning sites arejuxtaposed

SEQ ID NO:68: NTC9385C-intron RSM vector backbone. Bp 1 is start of ssiAupstream of ColE2 origin, last bp is end of polyadenylation signal. Exon2 encoded SalI (GTCGAC) and BglII (AGATCT) transgene cloning sites arejuxtaposed

SEQ ID NO:69: NTC9385C-intron RNAI vector backbone. Bp 1 is start ofssiA upstream of ColE2 origin, last bp is end of polyadenylation signal.Exon 2 encoded SalI (GTCGAC) and BglII (AGATCT) transgene cloning sitesare juxtaposed

SEQ ID NO:70: RSM-R6K gamma origin bacterial region. [NheI site-trpAterminator-R6K Origin-RSM-KpnI site]

SEQ ID NO:71: RNAI-R6K gamma origin bacterial region. [NheI site-trpAterminator-R6K Origin-RNAI-KpnI site]

SEQ ID NO:72: NTC9385R2 and NTC9385R2a intronic R6K gamma origin-RSMbacterial region. Sequence show is O1; O2 is reverse complement

SEQ ID NO:73: NTC9385R2 and NTC9385R2a intronic R6K gamma origin-RNAIbacterial region. Sequence show is O1; O2 is reverse complement

SEQ ID NO:74: RSM-ColE2 origin bacterial region. [NheI site-ssiA-ColE2Origin-RSM-KpnI site]

SEQ ID NO:75: RNAI-ColE2 origin bacterial region. [NheI site-ssiA-ColE2Origin-RNAI-KpnI site]

SEQ ID NO:76: NTC9385C2 and NTC9385C2a intronic C2 origin-RSM bacterialregion. Sequence show is O1; O2 is reverse complement

SEQ ID NO:77: NTC9385C2 and NTC9385C2a intronic C2 origin-RNAI bacterialregion. Sequence show is O1; O2 is reverse complement

Definition of Terms

A₄₀₅: Absorbance at 405 nanometers

AF: Antibiotic-free

APC: Antigen Processing Cell, for example, langerhans cells,plasmacytoid or conventional dendritic cells

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that is thesame or similar to a stated reference value

BAC: Bacterial artificial chromosome

Bacterial region: Region of a plasmid vector required for propagationand selection in the bacterial host

BE: Boundary element: Eukaryotic sequence that blocks the interactionbetween enhancers and promoters. Also referred to as insulator element.An example is the AT-rich unique region upstream of the CMV enhancer(XbaI to SpeI region; FIG. 1) that can function as an insulator/boundaryelement (Angulo A, Kerry D, Huang H, Borst E M, Razinsky A, Wu J et al.2000 J Virol 74: 2826-2839)

bp: basepairs

ccc: Covalently Closed Circular

cI: Lambda repressor

cITs857: Lambda repressor further incorporating a C to T (Ala to Thr)mutation that confers temperature sensitivity. cITs857 is a functionalrepressor at 28-30° C., but is mostly inactive at 37-42° C. Also calledcI857

Cm^(R): Chloramphenicol resistance

cmv: Cytomegalovirus

CMV promoter boundary element: AT-rich region of the humancytomegalovirus (CMV) genome between the UL127 open reading frame andthe major immediate-early (MIE) enhancer. Also referred to as uniqueregion (Angulo et al., Supra, 2000)

ColE2-P9 replication origin: a region which is specifically recognizedby the ColE2-P9 Rep protein to initiate DNA replication. Includes butnot limited to ColE2-P9 replication origin sequences disclosed in SEQ IDNO:13: ColE2 Origin (+7), SEQ ID NO:16: ColE2 Origin (+7, CpG free), SEQID NO:14: ColE2 Origin (Min) and SEQ ID NO:15: ColE2 Origin (core) andreplication functional mutations as disclosed in Yagura et al., 2006, JBacteriol 188:999 included herein by reference

ColE2 related replication origin: The ColE2-P9 origin is highlyconserved across the ColE2-related plasmid family. Fifteen ColE2 relatedplasmid members including ColE3 are compared in Hiraga et al., 1994, JBacteriol. 176:7233 and 53 ColE2 related plasmid members including ColE3are compared in Yagura et al., Supra, 2006. These sequences are includedherein by reference

ColE2-P9 plasmid: a circular duplex DNA molecule of about 7 kb that ismaintained at about 10 to 15 copies per host chromosome. The plasmidencodes an initiator protein (Rep protein), which is the onlyplasmid-specified trans-acting factor essential for ColE2-P9 plasmidreplication

ColE2-P9 replication origin RNA-OUT bacterial region: Contains aColE2-P9 replication origin for propagation and the RNA-OUT selectablemarker (e.g. SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25). Optionallyincludes a PAS, for example, the R6K plasmid CpG free ssiA primosomalassembly site (SEQ ID NO:17) or alternative ØX174 type or ABC typeprimosomal assembly sites, such as those disclosed in Nomura et al.,1991 Gene 108:15

ColE2 plasmid: NTC9385C, NTC9685C, NTC9385C2-O1, NTC9385C2-O2,NTC9385C2a-O1 and NTC9385C2a-O2 vectors, as well as modifications andalternative vectors containing a ColE2-P9 replication origin that weredisclosed in patent application PCT/US 13/00068 (Filing No. 61/743,219)entitled ‘DNA plasmids with improved expression’ and included herein byreference

delivery methods: Methods to deliver gene vectors [e.g.poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes,virosomes, chitosan, and other biodegradable polymers, electroporation,piezoelectric permeabilization, sonoporation, iontophoresis, ultrasound,corona plasma, plasma facilitated delivery, tissue tolerable plasma,laser microporation, shock wave energy, magnetic fields, contactlessmagneto-permeabilization, gene gun, microneedles, microdermabrasion,topical DNA application, naked DNA injection, hydrodynamic delivery,high pressure tail vein injection, needle free biojector, liposomes,microparticles, microspheres, nanoparticles, nanocapsules, virosomes,bacterial ghosts, bacteria, attenuated bacteria, etc] as known in theart and included herein by reference

DNA replicon: A genetic element that can replicate under its owncontrol; examples include plasmids, cosmids, bacterial artificialchromosomes (BACs), bacteriophages, viral vectors and hybrids thereof

E. coli: Escherichia coli, a gram negative bacteria

EGFP: Enhanced green fluorescent protein

EP: Electroporation

Eukaryotic expression vector: A vector for expression of mRNA, proteinantigens, protein therapeutics, shRNA, RNA or microRNA genes in a targeteukaryotic organism using RNA Polymerase I, II or III promoters

Eukaryotic replicative minicircle: a replicative minicircle eukaryoticexpression vector

Eukaryotic replicative pUC-free minicircle: a replicative minicircleeukaryotic expression vector that does not encode the pUC origin

Eukaryotic region: The region of a plasmid that encodes eukaryoticsequences and/or sequences required for plasmid function in the targetorganism. This includes the region of a plasmid vector required forexpression of one or more transgenes in the target organism includingRNA Pol II enhancers, promoters, transgenes and polyA sequences. Thisalso includes the region of a plasmid vector required for expression ofone or more transgenes in the target organism using RNA Pol I or RNA PolIII promoters, RNA Pol I or RNA Pol III expressed transgenes or RNAs.The eukaryotic region may optionally include other functional sequences,such as eukaryotic transcriptional terminators, supercoiling-induced DNAduplex destabilized (SIDD) structures, S/MARs, boundary elements, etc

Exon: A nucleotide sequence encoded by a gene that is transcribed andpresent within a mature mRNA product after RNA splicing to removeintrons has been completed

Expression vector: A vector for expression of mRNA, protein antigens,protein therapeutics, shRNA, RNA or microRNA genes in a target organism.

FU: Fluorescence units

g: Gram, kg for kilogram

gene of interest: gene to be expressed in the target organism. IncludesmRNA genes that encode protein or peptide antigens, protein or peptidetherapeutics, and mRNA, shRNA, RNA or microRNA that encode RNAtherapeutics, and mRNA, shRNA, RNA or microRNA that encode RNA vaccines,etc

Hr(s): Hour(s)

HTLV-I R: HTLV-I R 5′ untranslated region (UTR). Sequences andcompositions were disclosed in Williams, J A 2008 World PatentApplication WO2008153733 and included herein by reference

ID: Intradermal

IM: Intramuscular

immune response: Antigen reactive cellular (e.g. antigen reactive Tcells) or antibody (e.g. antigen reactive IgG) responses

IncB RNAI: plasmid pMU720 origin encoded RNAI (SEQ ID NO: 35) thatrepresses RNA II regulated targets (Wilson I W, Siemering K R, PraszkierJ, Pittard A J. 1997. J Bacteriol 179:742)

Intron: A nucleotide sequence encoded by a gene that is transcribed andsubsequently removed from a mature mRNA product by RNA splicing

kan: Kanamycin

kanR: Kanamycin Resistance gene

Kd: Kilodalton

kozak sequence: Optimized consensus DNA sequence gccRccATG (R=G or A)immediately upstream of an ATG start codon that ensures efficienttranlation initiation. A SalI site (GTCGAC) immediately upstream of theATG start codon (GTCGACATG) is an effective kozak sequence

minicircle: Covalently closed circular plasmid derivatives in which thebacterial region has been removed from the parent plasmid by in vivo orin vitro site specific recombination or in vitro restrictiondigestion/ligation. Minicircle vectors are replication incompetent inbacterial cells

mRNA: Messenger RNA

mSEAP: Murine secreted alkaline phosphatase

Nanoplasmid vector: Vector combining an RNA selectable marker with aR6K, ColE2 or ColE2 related replication origin. For example, NTC9385C,NTC9685C, NTC9385R, NTC9685R vectors and modifications disclosed inpatent application PCT/US 13/00068 (Filing No. 61/743,219) entitled ‘DNAplasmids with improved expression’ and included herein by reference andthe NTC9385C2 NTC9385C2a, NTC9385R2, NTC9385R2a, NTC9385R2b, NTC9385Ra,NTC9385RaF and NTC9385RbF replicative minicircle vectors of theinvention disclosed herein

NTC7382 promoter: A chimeric promoter comprising the CMV enhancer-CMVpromoter-HTLV R-synthetic rabbit β globin 3′ intron acceptor-exon 2-SRprotein binding site (three copies of GAAGAAGAC)-kozak sequence, with orwithout an upstream SV40 enhancer. The creation and application of thischimeric promoter is disclosed in Williams, Supra, 2008

NTC8385: NTC8385, NTC8485 and NTC8685 plasmids are antibiotic-freevectors that contain a short RNA (RNA-OUT) selectable marker instead ofan antibiotic resistance marker such as kanR. The creation andapplication of these RNA-OUT based antibiotic-free vectors are disclosedin Williams, Supra, 2008 and included herein by reference

NTC8485: NTC8485 is an antibiotic-free vector that contains a short RNA(RNA-OUT) selectable marker instead of an antibiotic resistance markersuch as kanR. The creation and application of NTC8485 is disclosed inWilliams, J A 2010 US Patent Application 20100184158 and included hereinby reference NTC8685: NTC8685 is an antibiotic-free vector that containsa short RNA (RNA-OUT) selectable marker instead of an antibioticresistance marker such as kanR. The creation and application of NTC8685is disclosed in Williams, Supra, 2010 and included herein by reference

NTC9385C: The NTC9385C vector disclosed in patent application PCT/US13/00068 (Filing No. 61/743,219) entitled ‘DNA plasmids with improvedexpression’ and included herein by reference has a spacer region encodedNheI-ssiA-ColE2 origin (+7) RNA-OUT-KpnI bacterial region (SEQ ID NO:23)linked through the flanking NheI and KpnI sites to the SEQ ID NO: 30eukaryotic region. Transgenes are cloned into NTC9385C between the SalIand BglII sites as described for the NTC9385P2, NTC9385P2a, NTC9385C2,NTC9385C2a, NTC9385R2, and NTC9385R2a vectors

NTC9385R: The NTC9385R vector disclosed in patent application PCT/US13/00068 (Filing No. 61/743,219) entitled ‘DNA plasmids with improvedexpression’ and included herein by reference has a spacer region encodedNheI-trpA terminator-R6K origin RNA-OUT-KpnI bacterial region (SEQ IDNO:26) linked through the flanking NheI and KpnI sites to the SEQ ID NO:30 eukaryotic region. Transgenes are cloned into NTC9385R between theSalI and BglII sites as described for the NTC9385P2, NTC9385P2a,NTC9385C2, NTC9385C2a, NTC9385R2, and NTC9385R2a vectors

OD₆₀₀: optical density at 600 nm

PAS: Primosomal assembly site. Priming of DNA synthesis on a singlestranded DNA ssi site. ØX174 type PAS: DNA hairpin sequence that bindspriA, which, in turn, recruits the remaining proteins to form thepreprimosome [priB, dnaT, recruits dnaB (delivered by dnaC)], which thenalso recruits primase (dnaG), which then, finally, makes a short RNAsubstrate for DNA polymerase I. ABC type PAS: DNA hairpin binds dnaA,recruits dnaB (delivered by dnaC) which then also recruits primase(dnaG), which then, finally, makes a short RNA substrate for DNApolymerase I. See Masai et al., 1990 J Biol Chem 265:15134. For example,the R6K plasmid CpG free ssiA primosomal assembly site (SEQ ID NO:17) oralternative ØX174 type or ABC type primosomal assembly sites, such asthose disclosed in Nomura et al., Supra, 1991

PAS-BH: Primosomal assembly site on the heavy (leading) strand

PAS-BH region: pBR322 origin region between ROP and PAS-BL(approximately pBR322 2067-2351)

PAS-BL: Primosomal assembly site on the light (lagging) strand

PBS: Phosphate buffered Saline

PCR: Polymerase Chain Reaction

pDNA: Plasmid DNA

pINT pR pL vector: The pINT pR pL integration expression vector isdisclosed in Luke et al., 2011 Mol Biotechnol 47:43 and included hereinby reference. The target gene to be expressed is cloned downstream ofthe pL promoter. The vector encodes the temperature inducible c1857repressor, allowing heat inducible target gene expression

P_(L) promoter: Lambda promoter left. P_(L) is a strong promoter that isrepressed by the cI repressor binding to OL1, OL2 and OL3 repressorbinding sites. The temperature sensitive c1857 repressor allows controlof gene expression by heat induction since at 30° C. the c1857 repressoris functional and it represses gene expression, but at 37-42° C. therepressor is inactivated so expression of the gene ensues

Plasmid: An extra chromosomal DNA molecule separate from the chromosomalDNA which is capable of replicating independently from the chromosomalDNA

pMB1 RNAI: pMB1 plasmid origin encoded RNAI (SEQ ID NO: 33) and RNAselectable marker (SEQ ID NO: 34) that represses RNAII regulated targetssuch as those described in Grabherr R, Pfaffenzeller I. 2006 US patentapplication US20060063232 and Cranenburgh R M. 2009; U.S. Pat. No.7,611,883

P_(min): Minimal 678 bp pUC replication origin SEQ ID NO:45 andfunctional variants with base substitutions and/or base deletions.Vectors described herein incorporating P_(min) includeNTC9385P2(0.85)-O1, NTC9385P2(0.85)-O2, NTC9385P2a(0.85)-O1, andNTC9385P2a(0.85)-02

Pol: Polymerase

polyA: Polyadenylation signal or site. Polyadenylation is the additionof a poly(A) tail to an RNA molecule. The polyadenylation signalcontains the sequence motif recognized by the RNA cleavage complex. Mosthuman polyadenylation signals contain an AAUAAA motif and conservedsequences 5′ and 3′ to it. Commonly utilized polyA signals are derivedfrom the rabbit β globin (NTC8485; FIG. 1), bovine growth hormone (gWIZ;pVAX1), SV40 early, or SV40 late polyA signals

pUC origin: pBR322-derived replication origin, with G to A transitionthat increases copy number at elevated temperature and deletion of theROP negative regulator

pUC free: Plasmid that does not contain the pUC origin. Non replicativefragments of the pUC origin may be included, for example the RNAIselectable marker (SEQ ID NO:34)

pUC plasmid: Plasmid containing the pUC origin

R6K plasmid: NTC9385R, NTC9685R, NTC9385R2-O1, NTC9385R2-O2,NTC9385R2a-O1, NTC9385R2a-O2, NTC9385R2b-O1, NTC9385R2b-O2,NTC9385Ra-O1, NTC9385Ra-O2, NTC9385RaF, and NTC9385RbF vectors as wellas modifications and alternative vectors containing a R6K replicationorigin that were disclosed in patent application PCT/US 13/00068 (FilingNo. 61/743,219) entitled ‘DNA plasmids with improved expression’ andincluded herein by reference. Alternative R6K vectors known in the artincluding, but not limited to, pCOR vectors (Gencell), pCpGfree vectors(Invivogen), and CpG free University of Oxford vectors including pGM169

R6K replication origin: a region which is specifically recognized by theR6K Rep protein to initiate DNA replication. Includes but not limited toR6K replication origin sequence disclosed as SEQ ID NO:11, and CpG freeversions (e.g. SEQ ID NO:12) as disclosed in Drocourt et al., U.S. Pat.No. 7,244,609 and incorporated herein by reference

R6K replication origin-RNA-OUT bacterial region: Contains a R6Kreplication origin for propagation and the RNA-OUT selectable marker(e.g. SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28)

Rep: Replication

Replicative minicircle: Covalently closed circular plasmid vector with ashort spacer region linking the 5′ and 3′ ends of the eukaryotic regionsequences in which the replication origin and/or the selection markerare encoded within an intron or 3′ UTR of a eukaryotic region or withinthe spacer region linking the 5′ and 3′ ends of the eukaryotic regionsequences. For dual eukaryotic region vectors, the replication originand/or the selectable marker may be cloned within an intron or 3′ UTR ofeither of the eukaryotic regions within the vector. In replicativeminicircle vectors of the invention, the spacer region preferably isless than 500 bp and may encode bacterial replication origins orselectable markers, bacterial transcription terminators, bacterialsupercoiling-induced DNA duplex destabilized (SIDD) structures. Inreplicative minicircle vectors of the invention, the spacer region mayoptionally encode eukaryotic sequences such as eukaryotic selectablemarkers, eukaryotic transcription terminators, supercoiling-induced DNAduplex destabilized (SIDD) structures, boundary elements, S/MARs, orother functionalities

Rep protein dependent plasmid: A plasmid in which replication isdependent on a replication (Rep) protein provided in Trans. For example,R6K replication origin, ColE2-P9 replication origin and ColE2 relatedreplication origin plasmids in which the Rep protein is expressed fromthe host strain genome. Numerous additional Rep protein dependentplasmids are known in the art, many of which are summarized in del Solaret al., 1998 Microbiol. Mol. Biol. Rev 62:434-464 which is includedherein by reference

RNA-IN: Insertion sequence 10 (IS10) encoded RNA-IN, an RNAcomplementary and antisense to a portion of RNA RNA-OUT. When RNA-IN iscloned in the untranslated leader of a mRNA, annealing of RNA-IN toRNA-OUT reduces translation of the gene encoded downstream of RNA-IN

RNA-IN regulated selectable marker: A genomically expressed RNA-INregulated selectable marker. In the presence of plasmid borne RNA-OUT(e.g. SEQ ID NO:21), expression of a protein encoded downstream ofRNA-IN is repressed. An RNA-IN regulated selectable marker is configuredsuch that RNA-IN regulates either 1) a protein that is lethal or toxicto said cell per se or by generating a toxic substance (e.g. SacB), or2) a repressor protein that is lethal or toxic to said bacterial cell byrepressing the transcription of a gene that is essential for growth ofsaid cell (e.g. murA essential gene regulated by RNA-IN tetR repressorgene). For example, genomically expressed RNA-IN-SacB cell lines forRNA-OUT plasmid propagation are disclosed in Williams, Supra, 2008 andincluded herein by reference. Alternative selection markers described inthe art may be substituted for SacB

RNA-OUT: Insertion sequence 10 (IS10) encoded RNA-OUT, an antisense RNAthat hybridizes to, and reduces translation of, the transposon geneexpressed downstream of RNA-IN. The sequence of the RNA-OUT RNA (SEQ IDNO:21) and complementary RNA-IN SacB genomically expressed RNA-IN-SacBcell lines can be modified to incorporate alternative functionalRNA-IN/RNA-OUT binding pairs such as those disclosed in Mutalik et al.,2012 Nat Chem Biol 8:447, including, but not limited to, the RNA-OUTA08/RNA-IN S49 pair, the RNA-OUT A08/RNA-IN S08 pair, and CpG freemodifications of RNA-OUT A08 that modify the CG in the RNA-OUT 5′ TTCGCSEQ ID NO: 21 sequence to a non-CpG sequence. An example of a CpG freeRNA-OUT selection marker, in which the two CpG motifs in the RNA-OUT RNA(one of which is present in the RNA-IN complementary region) areremoved, is given as SEQ ID NO:22. A multitude of alternativesubstitutions to remove the two CpG motifs (mutating each CpG to eitherCpA, CpC, CpT, ApG, GpG, or TpG) may be utilized to make a CpG freeRNA-OUT

RNA-OUT Selectable marker: An RNA-OUT selectable marker DNA fragmentincluding E. coli transcription promoter and terminator sequencesflanking an RNA-OUT RNA. An RNA-OUT selectable marker, utilizing theRNA-OUT promoter and terminator sequences, that is flanked by DraIII andKpnI restriction enzyme sites, and designer genomically expressedRNA-IN-SacB cell lines for RNA-OUT plasmid propagation, are disclosed inWilliams, Supra, 2008 (SEQ ID NO:20) and included herein by reference.The RNA-OUT promoter and terminator sequences flanking the RNA-OUT RNA(SEQ ID NO:21) may be replaced with heterologous promoter and terminatorsequences. For example, the RNA-OUT promoter may be substituted with aCpG free promoter known in the art, for example the I-EC2K promoter orthe P5/6 5/6 or P5/6 6/6 promoters disclosed in Williams, Supra, 2008and included herein by reference. An example of a CpG free RNA-OUTtranscription unit, in which the two CpG motifs in the RNA-OUT RNA (oneof which is present in the RNA-IN complementary region) and the two CpGmotifs in the RNA-OUT promoter are removed is given as SEQ ID NO: 22.The DraIII flanking restriction site contains a CpG, so CpG free RNA-OUTselectable markers are cloned with an alternative flanking restrictionsite, such as KpnI, BglII or EcoRI (flanking SEQ ID NO: 22). Vectorsincorporating the SEQ ID NO:22 CpG free RNA-OUT selectable marker may beselected for sucrose resistance using the RNA-IN-SacB cell lines forRNA-OUT plasmid propagation disclosed in Williams, Supra, 2008.Alternatively, the RNA-IN sequence in these cell lines can be modifiedto incorporate the 1 bp change needed to perfectly match the CpG freeRNA-OUT region complementary to RNA-IN as described in Example 1.

RNA polymerase I promoter: Promoter that recruits RNA Polymerase I tosynthesize ribosomal RNA

RNA polymerase II promoter: Promoter that recruits RNA Polymerase II tosynthesize mRNAs, most small nuclear RNAs and microRNAs. For example,constitutive promoters such as the human or murine CMV promoter,elongation factor 1 (EF1) promoter, the chicken β-actin promoter, theβ-actin promoter from other species, the elongation factor-1 α (EF1 α)promoter, the phosphoglycerokinase (PGK) promoter, the Rous sarcomavirus (RSV) promoter, the human serum albumin (SA) promoter, the α-1antitrypsin (AAT) promoter, the thyroxine binding globulin (TBG)promoter, the cytochrome P450 2E1 (CYP2E1) promoter, etc. The vectorsmay also utilize combination promoters such as the chicken β-actin/CMVenhancer (CAG) promoter, the human or murine CMV-derived enhancerelements combined with the elongation factor 1α (EF1α) promoters, CpGfree versions of the human or murine CMV-derived enhancer elementscombined with the elongation factor 1α (EF1α) promoters, the albuminpromoter combined with an α-fetoprotein MERII enhancer, etc, or thediversity of tissue specific or inducible promoters know in the art suchas the muscle specific promoters muscle creatine kinase (MCK), and C5-12or the liver-specific promoter apolipoprotein A-I (ApoAI), etc.

RNA polymerase III promoter: Promoter that recruits RNA Polymerase IIIto synthesize tRNAs, 5S ribosomal RNA, and other small RNAs. Forexample, Class I promoters such as the 5s rRNA promoter, Class IIpromoter such as tRNA promoters, Class III promoters such as the U6small nuclear RNA promoter or the H1 nuclear RNase P promoter, etc.

RNA selectable marker: An RNA selectable marker is a plasmid borneexpressed non translated RNA that regulates a chromosomally expressedtarget gene to afford selection. This may be a plasmid borne nonsensesuppressing tRNA that regulates a nonsense suppressible selectablechromosomal target as described by Crouzet J and Soubrier F 2005 U.S.Pat. No. 6,977,174 included herein by reference. This may also be aplasmid borne antisense repressor RNA, a non limiting list includedherein by reference includes RNA-OUT that represses RNA-IN regulatedtargets, pMB 1 plasmid origin encoded RNAI (SEQ ID NO: 33; a selectablemarker is given SEQ ID NO: 34) that represses RNAII regulated targets(Grabherr and Pfaffenzeller, Supra, 2006; Cranenburgh, Supra, 2009),IncB plasmid pMU720 origin encoded RNAI (SEQ ID NO: 35; a selectablemarker is given SEQ ID NO: 36) that represses RNA II regulated targets(SEQ ID NO: 37; Wilson et al., Supra, 1997), ParB locus Sok of plasmidR1 that represses Hok regulated targets, Flm locus FlmB of F plasmidthat represses flmA regulated targets (Morsey M A, 1999 U.S. Pat. No.5,922,583). An RNA selectable marker may be another natural antisenserepressor RNAs known in the art such as those described in Wagner E G H,Altuvia S, Romby P. 2002. Adv Genet 46:361 and Franch T, and Gerdes K.2000. Current Opin Microbiol 3:159. An RNA selectable marker may also bean engineered repressor RNAs such as synthetic small RNAs expressedSgrS, MicC or MicF scaffolds as described in Na D, Yoo S M, Chung H,Park H, Park J H, Lee S Y. 2013. Nat Biotechnol 31:170. An RNAselectable marker may also be an engineered repressor RNA such as SEQ IDNO: 38 as part of a selectable marker such as SEQ ID NO: 39 thatrepresses a target RNA such as SEQ ID NO: 40 fused to a target gene tobe regulated such as SacB in SEQ ID NO: 41 and SEQ ID NO:42

ROP: Repressor of primer

RSM: RNA selectable marker

SacB: Structural gene encoding Bacillus subtilis levansucrase.Expression of SacB in gram negative bacteria is toxic in the presence ofsucrose

SD: Standard deviation

SEAP: Secreted alkaline phosphatase

Selectable marker: A selectable marker, for example a kanamycinresistance gene or a RNA selectable marker

Selection marker: A selectable marker, for example a kanamycinresistance gene or a RNA selectable marker

SIDD: supercoiling-induced DNA duplex destabilized (SIDD) structures.These sites, when incorporated into a vector, may alter thesusceptibility of other sequences within the vector to be destabilized.This can alter function. For example, addition of a SIDD site to aexpression vector may reduce the helical destabilization of a promoter.This may increase or decrease promoter activity, depending on thepromoter since some promoters have increased expression with promoterhelical destabilization, while others will have reduced expression withpromoter helical destabilization

shRNA: Short hairpin RNA

S/MAR: Scaffold/matrix attached region. Eukaryotic sequences thatmediate DNA attachment to the nuclear matrix

SR: Spacer region.

Spacer region: As used herein, spacer region is the region linking the5′ and 3′ ends of the eukaryotic region sequences. The eukaryotic region5′ and 3′ ends are typically separated by the bacterial replicationorigin and bacterial selectable marker in plasmid vectors. In simplesingle RNA Pol II promoter vectors this will be between the RNA Pol IIpromoter region (5′ to either a promoter, enhancer, boundary element,S/MAR) and the RNA Pol II polyA region (3′ to either a polyA sequence,eukaryotic terminator sequence, boundary element, S/MAR). For example,in NTC8485 (FIG. 1) the 1492 bp spacer region is the region between NheIsite at 3737 and KpnI site at 1492. In dual RNA Pol II promoter vectors,the eukaryotic sequences separated by the spacer will depend on theorientation of the two transcription elements. For example, withdivergent or convergent RNA Pol II transcription units, the spacerregion may separate two polyA sequences or two enhancers respectively.In RNA Pol II promoter, RNA Pol III promoter dual expression vectors,the spacer region may separate an RNA Pol II enhancer and a RNA Pol IIIpromoter. In replicative minicircle vectors of the invention, thisspacer region preferably is less than 500 bp and may encode bacterialselectable markers, bacterial replication origins, bacterialtranscription terminators, bacterial supercoiling-induced DNA duplexdestabilized (SIDD) structures. In replicative minicircle vectors of theinvention, this spacer region may optionally encode eukaryotic sequencessuch as eukaryotic selectable markers, eukaryotic transcriptionterminators, supercoiling-induced DNA duplex destabilized (SIDD)structures, boundary elements, S/MARs, or other functionalities

ssi: Single stranded initiation sequences

SV40 enhancer: Simian Virus 40 genomic DNA that contains the 72 bp andoptionally the 21 bp enhancer repeats

target antigen: Immunogenic protein or peptide epitope, or combinationof proteins and epitopes, against which an immune response can bemounted. Target antigens may by derived from a pathogen for infectiousdisease or allergy applications, or derived from a host organism forapplications such as cancer, allergy, or autoimmune diseases. Targetantigens are well defined in the art. Some examples are disclosed inWilliams, Supra, 2008 and are included herein by reference

TE buffer: A solution containing approximately 10 mM Tris pH 8 and 1 mMEDTA

TetR: Tetracycline repressor

Transcription terminator: Bacterial: A DNA sequence that marks the endof a gene or operon for transcription. This may be an intrinsictranscription terminator or a Rho-dependent transcriptional terminator.For an intrinsic terminator, such as the trpA terminator, a hairpinstructure forms within the transcript that disrupts the mRNA-DNA-RNApolymerase ternary complex. Alternatively, Rho-dependent transcriptionalterminators require Rho factor, an RNA helicase protein complex, todisrupt the nascent mRNA-DNA-RNA polymerase ternary complex. Eukaryotic:PolyA signals are not ‘terminators’, instead internal cleavage at PolyAsites leaves an uncapped 5′ end on the 3′UTR RNA for nuclease digestion.Nuclease catches up to RNA Pol II and causes termination. Terminationcan be promoted within a short region of the poly A site by introductionof RNA Pol II pause sites (eukaryotic transcription terminator). Pausingof RNA Pol II allows the nuclease introduced into the 3′ UTR mRNA afterPolyA cleavage to catch up to RNA Pol II at the pause site. Anonlimiting list of eukaryotic transcription terminators know in the artinclude the C2×4 terminator (Ashfield R, Patel A J, Bossone S A, BrownH, Campbell R D, Marcu K B, Proudfoot N J. 1994. EMBO J 13:5656) and thegastrin terminator (Sato K, Ito R, Baek K H, Agarwal K, 1986. Mol. Cell.Biol. 6:1032). Eukaryotic transcription terminators may elevate mRNAlevels by enhancing proper 3′-end processing of mRNA (Kim D, Kim J D,Baek K, Yoon Y, Yoon J. 2003. Biotechnol Prog 19:1620)

Transgene: Gene of interest that is cloned into a vector for expressionin a target organism

ts: Temperature sensitive

μg: Microgram

μl: Microliter

UTR: Untranslated region of a mRNA (5′ or 3′ to the coding region)

VARNA: Adenoviral virus associated RNA, including VARNAI (VAI or VA1)and or VARNAII (VAII or VA2) from any Adenovirus serotype, for example,serotype 2, serotype 5 or hybrids thereof

VARNAI: Adenoviral virus associated RNAI, also referred to as VAI, orVA1, from any Adenovirus serotype, for example, serotype 2, serotype 5or hybrids thereof

Vector: A gene delivery vehicle, including viral (e.g. alphavirus,poxvirus, lentivirus, retrovirus, adenovirus, adenovirus related virus,etc) and nonviral (e.g. plasmid, MIDGE, transcriptionally active PCRfragment, minicircles, bacteriophage, etc) vectors. These are well knownin the art and are included herein by reference

Vector backbone: Eukaryotic region and bacterial region of a vector,without the transgene or target antigen coding region

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates generally to plasmid DNA vector methods andcompositions that improve plasmid manufacture and expression. Theinvention can be practiced to improve expression and manufacturing ofvectors such as eukaryotic expression plasmids useful for gene therapy,genetic immunization and or interferon therapy. Improved plasmidexpression is defined herein as improved transgene expression leveland/or expression duration in vitro or in vivo compared to a transgeneencoding pUC plasmid containing a spacer region encoded pUC replicationorigin. It is to be understood that all references cited herein areincorporated by reference in their entirety.

According to one preferred embodiment, the present invention providesfor method of increasing in vivo expression of transgene from covalentlyclosed super-coiled plasmid DNA, which comprises modifying the plasmidDNA to replace the pMB1, ColE1 or pBR322 derived replication origin andselectable marker with a replication origin selected from the groupconsisting of an ColE2-P9 replication origin, ColE2 related replicationorigin, and R6K replication origin and a RNA selectable marker;transforming the modified plasmid DNA into a Rep protein producingbacterial cell line rendered competent for transformation; and isolatingthe resultant transformed bacterial cells. The modified plasmid producedfrom these cells has increased transgene expression in the targetorganism.

In one preferred embodiment, the spacer region encoded pMB1, ColE1 orpBR322 derived replication origin is replaced with a CpG free ColE2origin to improve plasmid encoded transgene expression and manufacture.In another preferred embodiment, a primosome assembly site isincorporated into a ColE2 vector backbone to improve plasmid copynumber. In yet another preferred embodiment, the spacer region encodedpMB1, ColE1 or pBR322 derived replication origin is replaced with a CpGfree R6K origin to improve plasmid encoded transgene expression andmanufacture.

According to one preferred embodiment, the present invention providescompositions of short spacer region covalently closed super-coiledplasmid DNA eukaryotic vectors with improved transgene expression and E.coli manufacture, which comprises modifying the plasmid DNA to replacethe spacer region encoded replication origin in the vector spacer regionwith an intronic replication origin selected from the group consistingof an ColE2-P9 replication origin, ColE2 related replication origin, R6Kreplication origin, pUC replication origin and P_(min) pUC replicationorigin; transforming the modified plasmid DNA as necessary into a Repprotein producing bacterial cell line rendered competent fortransformation; and isolating the resultant transformed bacterial cells.The modified plasmid produced from these cells is a ‘replicativeminicircle’ vector with improved manufacture and transgene expression.

In one preferred embodiment, the vector spacer region encodedreplication origin is replaced with an intronic R6K replication originto improve plasmid encoded transgene expression and manufacture. Inanother preferred embodiment, the vector spacer region encodedreplication origin is replaced with an intronic pUC replication originto improve plasmid encoded transgene expression and manufacture. Inanother preferred embodiment, the vector spacer region encodedreplication origin is replaced with an intronic P_(min) pUC replicationorigin to improve plasmid encoded transgene expression and manufacture.In yet another preferred embodiment, the vector spacer region encodedreplication origin is replaced with an intronic ColE2 replication originto improve plasmid encoded transgene expression and manufacture. In yetanother preferred embodiment, the vector spacer region encodedreplication origin is replaced with an intronic CpG free ColE2replication origin to improve plasmid encoded transgene expression andmanufacture. In yet another preferred embodiment, the vector spacerregion encoded replication origin is replaced with an intronic CpG freeR6K replication origin to improve plasmid encoded transgene expressionand manufacture.

In yet another preferred embodiment, the vector spacer region encodedreplication origin is replaced with an 3′ UTR encoded R6K replicationorigin to improve plasmid encoded transgene expression and manufacture.In yet another preferred embodiment, the vector spacer region encodedreplication origin is replaced with an 3′ UTR encoded ColE2 replicationorigin to improve plasmid encoded transgene expression and manufacture.In yet another preferred embodiment, the vector spacer region encodedreplication origin is replaced with an 3′ UTR encoded CpG free ColE2replication origin to improve plasmid encoded transgene expression andmanufacture. In yet another preferred embodiment, the vector spacerregion encoded replication origin is replaced with an 3′ UTR encoded CpGfree R6K replication origin to improve plasmid encoded transgeneexpression and manufacture.

In yet another preferred embodiment, the vector spacer region encodedselectable marker is replaced with an 3′ UTR encoded RNA selectablemarker to improve plasmid encoded transgene expression and manufacture.In yet another preferred embodiment, the vector spacer region encodedselectable marker is replaced with an intron encoded RNA selectablemarker to improve plasmid encoded transgene expression and manufacture.

In yet another preferred embodiment, the vector spacer region directlylinks the eukaryotic region sequences that are typically separated bythe bacterial replication origin and bacterial selectable marker. In yetanother preferred embodiment, the vector eukaryotic regionpolyadenylation signal sequence is covalently linked directly to theenhancer of eukaryotic region promoter. In yet another preferredembodiment, a spacer region is included between the eukaryotic regionsequences that are typically separated by the bacterial replicationorigin and bacterial selectable marker. In yet another preferredembodiment the spacer region between the sequences that are typicallyseparated by the replication origin and selectable marker is 1 to 500bp. In yet another preferred embodiment the spacer region between thesequences that are typically separated by the replication origin andselectable marker encode bacterial or eukaryotic selectable markers,bacterial transcription terminators, eukaryotic transcriptionterminators, supercoiling-induced DNA duplex destabilized (SIDD)structures, boundary elements, S/MARs, RNA Pol I or RNA Pol IIIexpressed sequences or other functionalities. In yet another preferredembodiment, the spacer region is less than 500 bp and encodes an RNAselectable marker, with an R6K or ColE2 replication origin furtherencoded within an intron or a 3′ UTR. In yet another preferredembodiment, the spacer region is less than 500 bp and encodes an R6K orColE2 replication origin, with an RNA selectable marker further encodedwithin an intron or a 3′ UTR. In yet another preferred embodiment, thespacer region is less than 500 bp and encodes an RNA selectable marker,with an R6K or ColE2 replication origin further encoded within thespacer region.

The methods of plasmid modification of the present invention have beensurprisingly found to improve plasmid encoded transgene expression andmanufacture.

Plasmid encoded transgene expression is preferably improved by employingspecific constructs or compositions incorporated in a vector. Accordingto one preferred embodiment, the present invention provides acomposition for construction of a vector, comprising a RNA selectablemarker and a ColE2 origin with at least 90% sequence identity to thesequences set forth as SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, orSEQ ID NO: 16, and a plasmid DNA encoded eukaryotic region, wherein theColE2 origin is operably linked within an intron of the plasmid DNAencoded eukaryotic region. The RNA selectable marker may be operablylinked within an intron, a 3′ UTR or the spacer region. This novelvector configuration enables high yield manufacture of short spacerregion vectors. It has also been surprisingly found that this intronicColE2 origin improves plasmid encoded transgene expression. According toanother preferred embodiment, the eukaryotic region has at least 95%sequence identity to a sequence selected from the group consisting of:SEQ ID NO: 30, SEQ ID NO: 31.

According to another preferred embodiment, the present inventionprovides a composition for construction of a vector, comprising an RNAselectable marker and a R6K origin with at least 90% sequence identityto the sequences set forth as SEQ ID NO: 11, or SEQ ID NO: 12, and aplasmid DNA encoded eukaryotic region, wherein the R6K origin isoperably linked to an intron within the plasmid DNA encoded eukaryoticregion. The RNA selectable marker may be operably linked within anintron, a 3′ UTR or the spacer region. This novel vector configurationenables high yield manufacture of short spacer region vectors. It hasalso been surprisingly found that this intronic R6K origin improvesplasmid encoded transgene expression. According to another preferredembodiment, the eukaryotic region has at least 95% sequence identity toa sequence selected from the group consisting of: SEQ ID NO: 30, SEQ IDNO: 31.

According to another preferred embodiment, the present inventionprovides a composition for construction of a vector, comprising an RNAselectable marker and a pUC origin and a plasmid DNA encoded eukaryoticregion, wherein the pUC origin is operably linked to an intron withinthe plasmid DNA encoded eukaryotic region. The RNA selectable marker maybe operably linked within an intron, a 3′ UTR or the spacer region. Thisnovel vector configuration enables high yield manufacture of shortspacer region vectors. It has also been surprisingly found that thisintronic pUC origin improves plasmid encoded transgene expression.According to another preferred embodiment, the eukaryotic region has atleast 95% sequence identity to a sequence selected from the groupconsisting of: SEQ ID NO: 30, SEQ ID NO: 31.

According to another preferred embodiment, the present inventionprovides a composition for construction of a vector, comprising an RNAselectable marker and a P_(min) pUC origin with at least 90% sequenceidentity to the sequence set forth as SEQ ID NO: 45, and a plasmid DNAencoded eukaryotic region, wherein the P_(min) pUC origin is operablylinked to an intron within the plasmid DNA encoded eukaryotic region.The RNA selectable marker may be operably linked within an intron, a 3′UTR or the spacer region. This novel vector configuration enables highyield manufacture of short spacer region vectors. It has also beensurprisingly found that this intronic P_(min) pUC origin improvesplasmid encoded transgene expression. According to another preferredembodiment, the eukaryotic region has at least 95% sequence identity toa sequence selected from the group consisting of: SEQ ID NO: 30, SEQ IDNO: 31.

Plasmid encoded transgene expression is preferably improved by employingspecific constructs or compositions incorporated in a vector. Accordingto one preferred embodiment, the present invention provides acomposition for construction of a vector, comprising a ColE2 origin withat least 90% sequence identity to the sequences set forth as SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, and a plasmid DNAencoded eukaryotic region, wherein the ColE2 origin is operably linkedwithin a 3′ UTR of the plasmid DNA encoded eukaryotic region. An RNAselectable marker is incorporated into the vector either adjacent to thereplication origin or within an intron or within the spacer region.

According to another preferred embodiment, the present inventionprovides a composition for construction of a vector, comprising a 3′ UTRR6K origin with at least 90% sequence identity to the sequences setforth as SEQ ID NO: 11, or SEQ ID NO: 12, and a plasmid DNA encodedeukaryotic region, wherein the R6K origin is operably linked to a 3′ UTRwithin the plasmid DNA encoded eukaryotic region. An RNA selectablemarker is incorporated into the vector either adjacent to thereplication origin or within an intron or within the spacer region. Thisnovel vector configuration enables high yield manufacture of shortspacer region vectors.

The methods of plasmid modification of the present invention have beensurprisingly found to improve plasmid encoded transgene expression inthe target organism. Increased expression vectors may find applicationto improve the magnitude of DNA vaccination mediated antigen reactive Bor T cell responses for preventative or therapeutic vaccination,increase RNA and or protein transgene levels to improve gene replacementtherapy or gene knockdown therapy, increase plasmid based expressionlevels of DNA vector expressed therapeutic antibodies that neutralizeinfectious diseases such as influenza, HIV, malaria, hepatitis C virus,tuberculosis, etc.

The methods of plasmid modification of the present invention have beensurprisingly found to provide a solution to provide short spacer regionvectors with efficient high yield manufacture.

As used herein, the term “sequence identity” refers to the degree ofidentity between any given query sequence, e.g. SEQ ID NO: 2, and asubject sequence. A subject sequence may, for example, have at least 90percent, at least 95 percent, or at least 99 percent sequence identityto a given query sequence. To determine percent sequence identity, aquery sequence (e.g. a nucleic acid sequence) is aligned to one or moresubject sequences using any suitable sequence alignment program that iswell known in the art, for instance, the computer program ClustalW(version 1.83, default parameters), which allows alignments of nucleicacid sequences to be carried out across their entire length (globalalignment). Chema et al., 2003 Nucleic Acids Res., 31:3497-500. In apreferred method, the sequence alignment program (e.g. ClustalW)calculates the best match between a query and one or more subjectsequences, and aligns them so that identities, similarities, anddifferences can be determined. Gaps of one or more nucleotides can beinserted into a query sequence, a subject sequence, or both, to maximizesequence alignments. For fast pair-wise alignments of nucleic acidsequences, suitable default parameters can be selected that areappropriate for the particular alignment program. The output is asequence alignment that reflects the relationship between sequences. Tofurther determine percent identity of a subject nucleic acid sequence toa query sequence, the sequences are aligned using the alignment program,the number of identical matches in the alignment is divided by thelength of the query sequence, and the result is multiplied by 100. It isnoted that the percent identity value can be rounded to the nearesttenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to78.2.

Turning now to the drawings, FIG. 1. shows an annotated map of theantibiotic-free NTC8485 pUC origin expression vector with the locationsof the pUC origin, PAS-BH primosomal assembly site, SV40 enhancer, HpaIsite within the intron and other key elements indicated. The replicationorigin (PAS-BH and pUC origin) is from bp 32 to the DraIII (1345) site(1313 bp total). The antibiotic-free RNA-OUT selectable marker isbetween the DraIII (1345) and KpnI (1492) sites (147 bp total). Thebacterial region (trpA terminator, replication origin and RNA-OUTselectable marker=spacer region) of this vector is 1492 bp. Below themap an annotated sequence of the vector encoded HTLV-IR-Rabbit β globinhybrid intron (SEQ IND NO:1) is shown. The HTLV-I R derived 5′ intronicsplice donor region and the Rabbit β globin 3′ splice acceptor regionfunctionalities are separated by a HpaI site (GTTAAC, bold uppercase).The 5′ HTLV-I R derived splice donor (AGgtaagt; first 2 AG bases areexon 1) and rabbit β globin intron 1 derived 3′ splice acceptor (cagG;last G is exon 2) sites are double underlined. The 3′ splice acceptorpoly-pyrimidine tract (starting with cttttttct) is single underlined.This poly-pyrimidine tract sequence was altered from the native rabbit βglobin intron 1 sequence by replacing the native uppercase G and Aresidues in this region with t (ctGttttcA) to increase thepoly-pyrimidine tract consensus. The rabbit B globin 3′ acceptor branchsite (tgctgac) is single underlined. This intron is 225 bp and ispresent in the NTC8385, NTC8485, NTC8685, NTC9385C, NTC9685C, NTC9385R,and NTC9685R vectors.

FIG. 2 shows bioinformatics analysis of an intron containing the gWIZvector bacterial region (GBR2) encoded kanR selection marker-pUC origin.In this vector the kanR gene is antisense to the CMV promoter; theopposite sense orientation would be unacceptable due to safety concernsregarding the risk of kanR protein expression in the target organism.The kanR gene contains multiple cryptic splice acceptor and splice donorsites and potential sense and antisense promoters (not shown) predictedto interfere with intron function. The location and orientation of anexperimentally verified cryptic pUC origin promoter (Lemp N A, KiraokaK, Kasahara N, Logg C R. 2012. Nucleic Acids Res 40:7280) is shown(cryptic promoter). Splice signals were detected using the NetGene2(Brunak, S., Engelbrecht, J., and Knudsen, S. 1991 J Mol Biol 220,49-65) and Splicepredictor (Brendel, V., Xing, L. & Zhu, W. 2004.Bioinformatics 20, 1157-1169) programs while promoters were identifiedusing the Softberry (Mount Kisco, N.Y.) TSSG and FPROM programs

FIG. 3 shows bioinformatics analysis of introns containing the NTC9385P2and NTC9385P2a bacterial region encoded RNA-OUT selectable marker-pUCorigin in both orientations (P2-O1; P2-O2). A cryptic 209 bp exonderived from the pUC origin identified in A549 cells transfected withNTC9385P2-02 [and NTC9385P2(0.85)-O2] is indicated as well as thecryptic splice acceptor and cryptic splice donor used in this crypticexon. The location and orientation of an experimentally verified crypticpUC origin promoter (Lemp et al., Supra, 2012) is shown (crypticpromoter). Splice signals and promoters were detected as described inFIG. 2. The location of the regions removed in the NTC9385P2(0.85)-O1,NTC9385P2a(0.85)-O1, NTC9385P2(0.85)-O2 and NTC9385P2a(0.85)-O2 vectorsare indicated (0.85 region 1 and 0.85 region 2).

FIG. 4 shows an annotated map of the NTC9385P2a-O1-EGFP andNTC9385P2a-O2-EGFP intronic pUC origin-RNA-OUT replicative minicircleexpression vectors with the locations and orientations of the intronicRNA-OUT selectable marker, pUC replication origin (pUC origin) trpAterminator (SEQ ID NO: 29) and other key elements indicated. Thesevectors contain a 1436 bp intron.

FIG. 5 shows plasmid quality from intronic pUC origin-RNA-OUT expressionvectors NTC9385P2a-O1-EGFP, NTC9385P2a-O2-EGFP, NTC9385P2-O1-EGFP andNTC9385P2-O2-EGFP vectors versus a comparator spacer region encoded pUCorigin-RNA-OUT expression vector (NTC8385-EGFP). The top gel is a SYBRGreen I prestain, the bottom gel is after SYBR Green II poststaining for2 hrs followed by further electrophoresis to allow detection of shadowband or replication intermediates. SYBR Green I and II were obtainedfrom Invitrogen (Carlsbad, Calif., USA).

FIG. 6 depicts the NTC9385R2a-O1-EGFP and NTC9385R2a-O2-EGFP intronicR6K origin-RNA-OUT replicative minicircle expression vectors with thelocations and orientations of the intronic RNA-OUT selectable marker,R6K gamma replication origin (R6K mini-origin) trpA terminator (SEQ IDNO: 27) and other key elements indicated. These vectors contain a 685 bpintron.

FIG. 7 depicts the NTC9385C2a-O1-EGFP and NTC9385C2a-O2-EGFP intronicColE2 origin-RNA-OUT replicative minicircle expression vectors with thelocations and orientations of the intronic RNA-OUT selectable marker,ColE2-P9 replication origin (Replication origin) primosomal assemblysite (bacterial region is SEQ ID NO: 24) and other key elementsindicated. These vectors contain a 499 bp intron.

FIG. 8 shows plasmid quality from Table 7 fermentations of intronic R6Korigin-RNA-OUT expression vectors NTC9385R2-O1-EGFP, NTC9385R2-O2-EGFP,NTC9385R2a-O1-EGFP and NTC9385R2a-O2-EGFP vectors, versus a comparatorspacer region encoded R6K origin-RNA-OUT expression vector(NTC9385R-EGFP). The gel is a SYBR Green I prestain. No replicationintermediates or shadow band were detected after SYBR Green II poststainfor 2 hrs followed by further electrophoresis.

FIG. 9 depicts the NTC9385R2b-O2-EGFP intronic R6K origin-spacer regionRNA-OUT replicative minicircle expression vectors with the locations andorientations of the spacer region RNA-OUT selectable marker, intronicR6K gamma replication origin (R6K mini-origin SEQ ID NO: 11) trpAterminator and other key elements indicated. This vector contains a 539bp intron.

FIG. 10 shows pMB1 and ColE1 RNA I RNA selectable markers. The RNAIpromoter (−35 and −10) and RNAI antisense repressor RNA (italics; SEQ IDNO:33) is shown as well the location of the pUC high copy number G to Amutation (RNAI selectable marker: SEQ ID NO: 34)

FIG. 11 shows an IncB RNAI based RNA selectable marker. A) Genomicallyexpressed target of RNAI RNA selectable marker (SEQ ID NO: 37). Plasmidexpressed RNAI binding to the pseudoknot in the complementarygenomically expressed RNAII target prevents translation of thedownstream SacB gene, conferring sucrose resistance. The RNAI −10 and−35 promoter elements are mutated to prevent RNAI expression. B)Structure of plasmid expressed IncB RNAI RNA selectable marker (SEQ IDNO: 36) encoding the IncB RNAI antisense repressor (SEQ ID NO: 35).

FIG. 12 shows a designed synthetic CpG free RNA selectable marker. A)Structure of plasmid expressed engineered CpG free RSM antisense RNAmarker (SEQ ID NO: 39) encoding the CpG free antisense repressor RNA(SEQ ID NO: 38). B) Genomically expressed target of engineered CpG freeRNA selectable marker, RNA selection-sacB (RNAS). Plasmid expressedengineered CpG free RNA selectable marker binding to the complementarygenomically expressed RSM target (SEQ ID NO: 40) prevents translation ofthe downstream SacB gene, conferring sucrose resistance. Versions of RNAselection-SacB, in which the upstream promoter −35 and −10 promoterelements have either 5/6, 6/6 or 5/6, 5/6 or 5/6, 4/6 basepair match tothe TTGACA or TAATAT consensus sequences were made (SEQ ID NO:41; SEQ IDNO: 42) and cloned into the pINT integration vector to create pINT-RNAS(P5/6 6/6), pINT-RNAS (P5/6 4/6) (SEQ ID NO: 43) and pINT-RNAS (P5/65/6) (SEQ ID NO: 44).

FIG. 13 shows the NTC9385RbF-EGFP 3′ UTR R6K, intronic RNA-OUTreplicative minicircle expression vector. A) the locations andorientations of the intronic RNA-OUT selectable marker, 3′ UTR R6K gammaReplication origin (R6K mini-origin SEQ ID NO: 11) trpA terminator andother key elements indicated. This NTC9385RbF vector backbone (with EGFPexcised, SalI and BglII transgene cloning sites juxtaposed) is presentedas SEQ ID NO: 47. An alternative vector backbone, NTC9385RbF-RSM (withEGFP excised, SalI and BglII transgene cloning sites juxtaposed), inwhich the RNA-OUT selectable marker is substituted with the CpG free RSMantisense RNA marker (SEQ ID NO: 39) is presented as SEQ ID NO: 48. Analternative vector backbone, NTC9385RbF-RNAI (with EGFP excised, SalIand BglII transgene cloning sites juxtaposed), in which the RNA-OUTselectable marker is substituted with the RNAI selectable marker (SEQ IDNO: 34) is presented as SEQ ID NO: 49. B) Annotated map of theNTC9385RbF 3′ UTR and polyA region. Open reading frames (cutoff ofminimum 20 amino acids) on the complementary strand are indicated witharrowed lines. No open reading frames are present in the senseorientation.

The invention also relates to compositions and methods for producingshort spacer region replicative minicircle plasmids with dramaticallyimproved manufacturing yields and simplified manufacturing compared toalternative short spacer region vectors such as minicircles. The presentinvention also provides sequences that, when introduced into a vectorbackbone, increase plasmid encoded transgene expression.

The invention also relates to compositions and methods for producinghigh expression level plasmids. The present invention provides sequencesthat, when introduced into a vector backbone, increase plasmid encodedtransgene expression.

The surprising observation that a ColE2 replication origin-RNA selectionmarker or R6K replication origin-RNA selectable marker can be utilizedas a plasmid encoded transgene expression enhancer is disclosed.

As described herein, plasmid encoded transgene expression is increasedby replacement of the pMB1, ColE1 or pBR322 derived origin-selectionmarker bacterial region with an R6K origin-RNA selectable marker in theplasmid spacer region. In yet another preferred embodiment, the R6Korigin is CpG free. In yet another preferred embodiment, the R6K originis included with an RNA-OUT selectable marker.

In yet another preferred embodiment, plasmid encoded transgeneexpression is increased by replacement of the pMB1, ColE1 or pBR322derived origin-selection marker bacterial region with a ColE2 origin-RNAselectable marker in the plasmid spacer region. In yet another preferredembodiment, the ColE2 origin is CpG free. In yet another preferredembodiment, the ColE2 origin is included with an RNA-OUT selectablemarker. In yet another preferred embodiment, the ColE2 origin isincluded with a primosome assembly site.

The surprising observation that a ColE2, R6K, pUC or P_(min) pUCreplication origin can be inserted into an intron of a eukaryotic RNAPol II transcription unit without decreasing intron efficiency ortransgene expression is disclosed.

The surprising observation that a ColE2 or R6K, replication origin canbe inserted into an 3′ UTR of a eukaryotic RNA Pol II transcription unitwithout decreasing transgene expression is disclosed.

As described herein, plasmid encoded transgene expression is improved byreplacement of the vector spacer region encoded replication origin withan R6K origin in an intron or 3′ UTR of a eukaryotic RNA Pol IItranscription unit. In yet another preferred embodiment, the R6K originis CpG free. In yet another preferred embodiment, the R6K origin isincluded with an RNA-OUT selectable marker.

In yet another preferred embodiment, plasmid encoded transgeneexpression is improved by replacement of the vector spacer regionencoded replication origin with a ColE2 origin in an intron or 3′ UTR ofa eukaryotic RNA Pol II transcription unit. In yet another preferredembodiment, the ColE2 origin is CpG free. In yet another preferredembodiment, the ColE2 origin is included with an RNA-OUT selectablemarker. In yet another preferred embodiment, the ColE2 origin isincluded with a primosome assembly site.

In yet another preferred embodiment, plasmid encoded transgeneexpression is improved by replacement of the vector spacer regionencoded replication origin with a pUC origin in an intron of aeukaryotic RNA Pol II transcription unit. In yet another preferredembodiment, the pUC origin is included with an RNA-OUT selectablemarker.

In yet another preferred embodiment, plasmid encoded transgeneexpression is improved by replacement of the vector spacer regionencoded replication origin with a P_(min) pUC origin in an intron of aeukaryotic RNA Pol II transcription unit. In yet another preferredembodiment, the P_(min) pUC origin is included with an RNA-OUTselectable marker.

Improved plasmid encoded transgene expression is defined herein asimproved transgene expression level and/or expression duration in vitroor in vivo compared to a transgene encoding pUC plasmid containing aspacer region encoded pUC replication origin.

EXAMPLES

The methods of the invention are further illustrated by the followingexamples. These are provided by way of illustration and are not intendedin any way to limit the scope of the invention.

Example 1: pUC, R6K and ColE2 Replication Origin Plasmid Replication andProduction

pUC Origin Vector Replication and Production Background:

The vast majority of therapeutic plasmids use the pUC origin which is ahigh copy derivative of the pMB1 origin (closely related to the ColE1origin). For pMB1 replication, plasmid DNA synthesis is unidirectionaland does not require a plasmid borne initiator protein. The pUC originis a copy up derivative of the pMB1 origin that deletes the accessoryROP (rom) protein and has an additional temperature sensitive mutationthat destabilizes the RNAI/RNAII interaction. Shifting of a culturecontaining these origins from 30 to 42° C. leads to an increase inplasmid copy number. pUC plasmids can be produced in a multitude of E.coli cell lines. pUC plasmid propagation and fermentations reportedherein were performed using cell line NTC48165=DH5α dcm atλ::P5/66/6-RNA-IN-SacB or NTC54208=XL1Blue dcm atλ::P5/6 6/6-RNA-IN-SacB thecreation of which are disclosed in Carnes A E, Luke J M, Vincent J M,Schukar A, Anderson S, Hodgson C P, and Williams J A. 2011 BiotechnolBioeng 108:354-363.

R6K Origin Vector Replication and Production Background:

The R6K gamma plasmid replication origin requires a single plasmidreplication protein π that binds as a replication initiating monomer tomultiple repeated ‘iteron’ sites (seven core repeats containing TGAGNGconsensus) and as a replication inhibiting dimer to repressive sites(TGAGNG) and to iterons with reduced affinity. Replication requiresmultiple host factors including IHF, DnaA, and primosomal assemblyproteins DnaB, DnaC, DnaG (Abhyankar et al., 2003 J Biol Chem278:45476-45484). The R6K core origin contains binding sites for DnaAand IHF that affect plasmid replication since π, IHF and DnaA interactto initiate replication.

Different versions of the R6K gamma replication origin have beenutilized in various eukaryotic expression vectors, for example pCORvectors (Soubrier et al., 1999, Gene Therapy 6:1482) and a CpG freeversion in pCpGfree vectors (Invivogen, San Diego Calif.), and pGM169(University of Oxford). Incorporation of the R6K replication origin perse does not improve transgene expression levels compared to an optimizedpUC origin vector (Soubrier et al., Supra, 1999). However, use of aconditional replication origin such as R6K gamma that requires aspecialized cell line for propagation adds a safety margin since thevector will not replicate if transferred to a patient's endogenousflora.

A highly minimalized R6K gamma derived replication origin (SEQ ID NO:11)that contains core sequences required for replication (including theDnaA box and stb 1-3 sites; Wu et al., 1995. J Bacteriol. 177:6338-6345), but with the upstream π dimer repressor binding sites anddownstream π promoter deleted (by removing one copy of the iterons) wasdisclosed in patent application PCT/US 13/00068 (Filing No. 61/743,219)entitled ‘DNA plasmids with improved expression’ and included herein byreference. The NTC9385R vector backbone (SEQ ID NO: 62) including thisminimalized R6K origin and the RNA-OUT AF selectable marker in thespacer region, was disclosed in patent application PCT/US 13/00068(Filing No. 61/743,219) entitled ‘DNA plasmids with improved expression’and included herein by reference.

Typical R6K production strains express from the genome the π proteinderivative PIR116 that contains a P106L substitution that increases copynumber (by reducing π dimerization; π monomers activate while π dimersrepress). Fermentation results with pCOR (Soubrier et al., Supra, 1999)and pCpG plasmids (Hebel H L, Cai Y, Davies L A, Hyde S C, Pringle I A,Gill D R. 2008. Mol Ther 16: 5110) were low, around 100 mg/L in PIR116cell lines.

Mutagenesis of the pir-116 replication protein and selection forincreased copy number has been used to make new production strains. Forexample, the TEX2pir42 strain contains a combination of P106L and P42L.The P42L mutation interferes with DNA looping replication repression.The TEX2pir42 cell line improved copy number and fermentation yieldswith pCOR plasmids with reported yields of 205 mg/L (Soubrier F.Circular DNA molecule having a conditional origin of replication,process for their preparation and their use in gene therapy. WorldPatent Application WO2004033664. 2004).

Other combinations of π copy number mutants that improve copy numberinclude ‘P42L and P113S’ and ‘P42L, P106L and F107S’ (Abhyankar et al.,2004. J Biol Chem 279:6711-6719).

RNA-OUT selectable marker-R6K plasmid propagation and fermentationsreported herein were performed using heat inducible ‘P42L, P106L andF107S’ π copy number mutant cell line NTC711231, the creation of whichis disclosed in patent application PCT/US 13/00068 (Filing No.61/743,219) entitled ‘DNA plasmids with improved expression’ andincluded herein by reference. NTC711231=NTC54208-pR pL (OL1-G to T)P42L-P106L-F107S (P3-). NTC54208=XL1Blue dcm attλ::P5/6 6/6-RNA-IN-SacB.Fermentations were additionally performed in the equivalent DH5α hoststrain NTC711772=NTC48165-pR pL (OL1-G to T) P42L-P106L-F107S (P3-).NTC48165=DH5α dcm attλ::P5/6 6/6-RNA-IN-SacB. Fermentations were alsoperformed using host strain NTC731871, a ‘P42L, P113S’ π copy numbermutant cell line which is equivalent to NTC711231 except for use of thealternative ‘P42L, P113S’ π copy number mutant disclosed in patentapplication PCT/US 13/00068 (Filing No. 61/743,219) entitled ‘DNAplasmids with improved expression’.

ColE2 origin vector replication and production background: The ColE2replication origin (for example, ColE2-P9) is highly conserved acrossthe ColE2-related plasmid family. Fifteen members are compared in Hiragaet al., Supra, 1994, and fifty three ColE2 related plasmid membersincluding ColE3 are compared in Yagura et al., Supra, both referencesare included herein by reference. Plasmids containing this origin arenormally 10 copies/cell (low copy number). For gene therapy vectorapplication, ColE2 replication origin vector copy number needs to beimproved dramatically.

Expression of the ColE2-P9 replication (Rep) protein is regulated byantisense RNA (RNAI). Copy number mutations have been identified thatinterfere with this regulation and raise the copy number to 40/cell(Takechi et al., 1994 Mol Gen Genet 244:49-56).

RNA-OUT selectable marker-ColE2 plasmid propagation and fermentationswere performed using heat inducible ‘G194D’ Rep protein copy numbermutant cell line NTC710351 the creation of which is disclosed in patentapplication PCT/US 13/00068 (Filing No. 61/743,219) entitled ‘DNAplasmids with improved expression’ and included herein by reference.

The following vectors including NTC9385C (SEQ ID NO: 63) containing theminimal ColE2-P9 origin (Yagura and Itoh 2006 Biochem Biophys Res Commun345:872-877) and various origin region modifications were disclosed inpatent application PCT/US 13/00068 (Filing No. 61/743,219) entitled ‘DNAplasmids with improved expression’ and included herein by reference.

+7-ssiA:

This combines the ColE2 origin (+7) (SEQ ID NO:13) with ssiA fromplasmid R6K (SEQ ID NO:17). Thus ssiA vectors contain, in addition tothe ColE2-P9 origin, a downstream primosome assembly site. Like mostplasmid origins, the ColE2 origin contains a primosomal assembly siteabout 100 bp downstream of the origin (Nomura et al., Supra, 1991). Thissite primes lagging strand DNA replication (Masai et al., 1990 J BiolChem 265:15124-15133) which may improve plasmid copy number or plasmidquality. The ColE2 PAS (ssiA) is similar to PAS-BH (ColE1 ssiA=PAS-BLMarians et al., 1982 J Biol Chem 257:5656-5662) and both sites (andPAS-BH) are CpG rich ØX174 type PAS. A CpG free PAS (ssiA from R6K;Nomura et al., Supra, 1991; SEQ ID NO:17) that acts as a dnaA, dnaB dnaC(ABC) primosome on a dnaA box hairpin sequence (Masai et al., Supra,1990) was selected for inclusion in the +7-ssiA vectors. Alternative ABCor ØX174 type PAS sequences are functionally equivalent to ssiA fromR6K, and may be substituted for ssiA in these ColE2 replication originvectors. NTC9385C incorporates the +7-ssiA origin region.

+7 CpG Free-ssiA (SEQ ID NO:18):

This combines the ColE2 replication origin (+7 CpG free) (SEQ ID NO:16)with ssiA from plasmid R6K (SEQ ID NO:17). The single CpG in the ColE2replication origin was removed from the vector by site directedmutagenesis. A version with flanking SphI and KpnI restriction sites forcloning is disclosed as SEQ ID NO: 19.

Yagura et al., Supra, 2006 have demonstrated that the Min ColE2Replication origin (SEQ ID NO:14, which is reverse complement ofresidues 7-38, in FIG. 1 of Yagura et al., Supra, 2006) can be furtherdeleted without eliminating replication function. Yagura et al., Supra,2006, demonstrated that the core sequence is residues 8-35, withresidues 5-36 are required for full activity. The +7 ColE2 Replicationorigin (SEQ ID NO:13; which is the reverse complement of residues 0-44in FIG. 1 of Yagura et al., Supra, 2006) could therefore be reduced tospan residues 8-35 or 5-36 of FIG. 1 of Yagura et al., Supra, 2006 (SEQID NO:15). Such vectors should replicate similarly to the disclosedvectors. As well, a number of base changes can be made within the coreColE2 origin 8-34 region that do not affect ColE2 replication (seechanges to residues that retain function in Table 2; Yagura et al.,Supra, 2006).

The +7(CpG free)-ssiA ColE2 origin (SEQ ID NO 18) or +7(CpG free) ColE2origin (SEQ ID NO 16) are smaller CpG free replication originalternatives to the 260 bp CpG free R6K replication origins (SEQ IDNO:12). CpG free ColE2 origins may be utilized to construct CpG freeplasmid vectors. Combinations of a CpG free ColE2 or R6K replicationorigin with a CpG free RNA-OUT selectable marker (SEQ ID NO: 22) may beutilized to construct antibiotic-free CpG free bacterial regions for CpGfree plasmid vectors (e.g. SEQ ID NO:25; SEQ ID NO:28). A CpG freeR6K-RNA-OUT bacterial region was created by replacement of the kanRmarker in a CpG free R6K replication origin (SEQ ID NO:12) vector with aCpG free RNA-OUT selectable marker (KpnI—SEQ ID NO: 22—BglII restrictionfragment). This CpG free RNA-OUT selectable marker encoding R6K originvector was successfully recovered as sucrose resistant colonies aftertransformation of R6K production host cell line NTC711231. This vectordirectly links the reverse complement of CpG free R6K replication origin(SEQ ID NO:12) through the KpnI site to the CpG free RNA-OUT selectablemarker (SEQ ID NO: 22) in the orientation R6K origin>RNA-OUT> as opposedto the divergent orientation <R6K origin RNA-OUT> composition disclosedas SEQ ID NO:28. Expression of an EGFP transgene expressing version ofthis vector in A549 cells transfected as described in Example 2 wasimproved at 48 hrs post transfection compared to the kanR parent vector(4657±593 FU versus 3573±388 FU with the kanR parent vector). Thisdemonstrates that various orientations of RNA-OUT and the replicationorigin may be utilized to create replicative minicircle vectors withimproved expression, and that alternative RNA selectable markers can beutilized. Further, a CpG free ColE2-RNA bacterial region was created byreplacement of the RNA-OUT marker and R6K origin in the CpG free R6Kreplication origin vector above with the +7(CpG free)-ssiA ColE2 origin(SEQ ID NO 19)-CpG free RNA-OUT selectable marker (SEQ ID NO: 22) as aSphI BglII restriction fragment (SEQ ID NO: 25). This CpG free RNA-OUTselectable marker encoding ColE2 origin vector was successfullyrecovered as sucrose resistant colonies after transformation of ColE2production host cell line NTC710351. Expression of an EGFP transgeneexpressing version of this CpG free ColE2 origin-RNA-OUT vector in A549cells transfected as described in Example 2 was improved at 48 hrs posttransfection compared to the CpG free R6K-RNA-OUT-vector described above(1921±123 FU with the CpG free ColE2 origin-RNA-OUT vector versus1579±207 FU with the CpG free R6K origin-RNA-OUT vector). The successfulconstruction of these disclosed vectors demonstrate that CpG free ColE2or R6K replication origins can be combined with a CpG free RNA-OUTselectable marker to construct antibiotic-free CpG free bacterialregions for CpG free plasmid vectors. The cell lines for selection (e.g.NTC711231, NTC711772, or NTC731871 for R6K and NTC710351 for ColE2) maybe modified to alter the RNA-IN sequence in attλ::P5/6 6/6-RNA-IN-SacBto match the CpG free RNA-OUT encoded single base change that removesthe CpG motif in the RNA-OUT RNA that is present in the RNA-INcomplementary region (CpG free RNAIN). For example, robust sucroseselection and high plasmid yields and quality were obtained aftertransformation of CpG free RNA-OUT selectable marker (SEQ ID NO: 22) CpGfree R6K origin (SEQ ID NO: 12) vectors into R6K production hostNTC791342=XL1Blue dcm attλ::P5/6 6/6-CpG free RNA-IN-SacB-pR pL (OL1-Gto T) P42L-P106L-F107S (P3-) which incorporates the one bp change inRNA-IN needed to perfectly complement the CpG free RNA-OUT. For example,CpG free RNA-OUT selectable marker (SEQ ID NO: 22) -CpG free ColE2origin-CpG free ssiA (SEQ ID NO: 18) (incorporating the CpG free ColE2RNA-OUT bacterial region; SEQ ID NO: 25) vectors may be propagated inColE2 production host NTC791381=XL1Blue dcm attλ::P5/6 6/6-CpG freeRNA-IN-SacB-pR pL (OL1-G to T) ColE2rep G194D which incorporates the onebp change in RNA-IN needed to perfectly complement the CpG free RNA-OUT.

An alternative CpG free RNA selectable marker that may be substitutedfor the CpG free RNA-OUT selectable marker in the creation of CpG freeplasmid vectors is the RSM selectable marker (SEQ ID NO: 39) withflanking CpG free restriction sites (e.g. KpnI, BglII) replacing the CpGcontaining DraIII restriction site. Alternatively, the RSM antisenserepressor RNA (SEQ ID NO: 38) with flanking CpG free promoter andterminator sequences could be substituted for the CpG free RNA-OUTselectable marker in the creation of CpG free plasmid vectors.

Use of a conditional replication origin such as these ColE2 origins thatrequires a specialized cell line for propagation adds a safety marginsince the vector will not replicate if transferred to a patientsendogenous flora.

Example 2: NTC9385R and NTC9385C Vector Construction, Manufacture andExpression

The NTC9385C and NTC9385R AF eukaryotic expression vectors incorporatingnovel ColE2-P9 or R6K derived vector origins, respectively were made. Toreplace the spacer region encoded pUC origin with a ColE2 origin, theColE2 origin (+7) (SEQ ID NO:13) combined with ssiA from plasmid R6K(SEQ ID NO:17) from Example 1 was used to make NTC9385C (SEQ ID NO: 63).To replace the spacer region encoded pUC origin with a R6K origin theR6K origin (SEQ ID NO:11) from Example 1 was used to make NTC9385R (SEQID NO: 62). The R6K gamma origin vector was constructed by swapping inthe R6K gamma origin (SEQ ID NO:1) in a NotI-DraIII R6K origin syntheticgene for the corresponding NotI-DraIII pUC origin region. The ColE2origin vector was constructed in a similar fashion, by swapping in the+7 ssiA ColE2 origin in a NheI-DraIII synthetic gene for thecorresponding NheI-DraIII pUC origin region.

The 466 bp Bacterial region [NheI site-trpA terminator-R6KOrigin-RNA-OUT-KpnI site] for NTC9385R and NTC9685R is shown in SEQ IDNO:26. The 281 bp Bacterial region [NheI site-ssiA-ColE2 Origin(+7)-RNA-OUT-KpnI site] for NTC9385C and NTC9685C is shown in SEQ IDNO:23.

High fermentation yields in HyperGRO media are obtained with thesevectors. For example 392 mg/mL with NTC9385R-EGFP in R6K production cellline NTC711231 and 672 mg/L with NTC9385C-EGFP in ColE2 production cellline NTC710351 (Table 7).

These are just a few possible nonlimiting short spacer region vectorconfigurations. Many alternative vector configurations incorporating thenovel R6K or ColE2 origin vector modifications may also be made,including but not limited to vectors with alternative selection markers,alternative promoters, alternative terminators, and differentorientations of the various vector-encoded elements or alternative R6Kor ColE2 origins as described in Examples 1 to 11.

An example strategy for cloning into these vectors is outlined below.

GTCGAC ATG--------Gene of interest----Stop codon------AGATCTSalI                                                 BglII

For the NTC9385C and NTC9385R vectors, the ATG start codon (doubleunderlined) is immediately preceded by a unique SalI site. The SalI siteis an effective Kozak sequence for translational initiation.

EGFP and muSEAP transgene versions of NTC9385C and NTC9385R wereconstructed by standard restriction fragment swaps. The muSEAP gene issecreted using its endogenous secretion signal, while EGFP is cellassociated. Expression levels in vitro were determined using EGFP, whileexpression levels in vivo were determined using muSEAP. Expressionlevels were compared to the NTC8685 vector, the gWIZ vector, and aminicircle comparator. NTC8685 and gWIZ are examples of vectorscomprising a spacer region greater than 500 basepairs.

Adherent HEK293 (human embryonic kidney) and A549 (human lungcarcinoma),cell lines were obtained from the American Type CultureCollection (Manassas, Va., USA). Cell lines were propagated inDulbecco's modified Eagle's medium/F12 containing 10% fetal bovine serumand split (0.25% trypsin-EDTA) using Invitrogen (Carlsbad, Calif., USA)reagents and conventional methodologies. For transfections, cells wereplated on 24-well tissue culture dishes, plasmids were transfected intocell lines using Lipofectamine 2000 following the manufacturer'sinstructions (Invitrogen).

Total cellular lysates for EGFP determination were prepared byresuspending cells in cell lysis buffer (BD Biosciences Pharmingen, SanDiego, Calif., USA), lysing cells by incubating for 30 min at 37° C.,followed by a freeze-thaw cycle at −80° C. Lysed cells were clarified bycentrifugation and the supernatants assayed for EGFP by FLX800microplate fluorescence reader (Bio-Tek, Winooski, Vt., USA). Theresults are summarized in Tables 3 and 8.

Groups of five mice were injected with plasmid DNA in an IACUC-approvedstudy. Five micrograms of muSEAP plasmid in 25 or 50 μL ofphosphate-buffered saline (PBS) was injected intramuscularly (IM) into atibialis cranialis muscles of female BALB/c mice or ND4 Swiss Webstermice (8 to 10 weeks old) followed by Ichor TriGrid electroporation. SEAPlevels in serum were determined using the Phospha-light SEAP ReporterGene Assay System from Applied Biosystems (Foster City, Calif.)according to the manufacturer's instructions. The results are summarizedbelow.

The NTC9385C and NTC9385R vectors had similar expression to the NTC8685vector in vitro, and higher expression than the gWIZ comparator (Table1). Thus substitution of the R6K or ColE2 replication origin for the pUCorigin in the spacer region was not detrimental for eukaryotic cellexpression. However, surprisingly, in vivo expression was dramaticallyimproved compared to NTC8685 or gWIZ with the ColE2 and R6K originvectors (Table 1). For example the NTC9385C vector was unexpectedlyimproved 1.5 to 3.8× that of NTC8385 or NTC8685 (Table 1) after IMdelivery with EP.

TABLE 1 gWIZ, NTC9385C and NTC9385R Nanoplasmid expression compared toNTC8685 % NTC8685 % NTC8685 % NTC8685 % NTC8685 % NTC8685 expressionexpression expression expression expression T = 7 days T = 7 days T = 28days T = 28 days Plasmid in vitro ^(a) BALB/c ^(b) ND4 ^(b) BALB/c ^(b)ND4 ^(b) gWIZ 58 59  57 21  57 NTC8385 NA NA 101 NA 101 NTC9385C 92 377 349 150  233 NTC9385R NA NA 154 NA 216 Minicircle ^(c) NA 89 NA 40 NA^(a) 100 ng/well EGFP transgene vectors transfected with lipofectamineinto HEK293 cells ^(b) murine SEAP (muSEAP) transgene vectors in 8-10week old BALB/c or ND4 Swiss Webster female mice, 5 μg dose with EPintramuscular into one anterior tibialis muscle followed by IchorTriGrid electroporation. 25 μL dose for ND4 mice, 50 μL dose for BALB/c.^(c) Minicircle equivalent to NTC9385C or NTC9385R, with NheI-KpnIregion containing the replication origin and RNA-OUT selectable marker(bacterial region) removed from NTC8385-muSEAP by SpeI/NheI digestion,gel purification of the eukaryotic region, in vitro ligation andsupercoiling with DNA gyrase. The SpeI site is the same site used totruncate the CMV promoter to make NTC8685, NTC9385C and NTC9385R vectorsso the minicircle eukaryotic region is the same as NTC9385C-muSEAP andNTC9385R-muSEAP, the difference being the C2 and RNA-OUT regionincluding the KpnI site is deleted in the minicircle. NA = Not assayed

This improved in vivo expression was not specific to the CMV promoter.Versions of NTC8685-muSEAP and NTC9385C-muSEAP were constructed in whichthe murine creatine kinase (MCK) promoter (3 copies of the MCK Enhancerupstream of the MCK promoter and 50 bp of the MCK exon 1 leadersequence; Wang B, Li J, Fu F H, Chen C, Zhu X, Zhou L, Jiang X, Xiao X.2008. Gene Ther 15:1489) was substituted for the CMV promoter. The swapsreplaced the entire CMV enhancer CMV promoter-exon 1 leader (NTC8685:from a XbaI site immediately after the SV40 enhancer to a SacII site inthe CMV derived exon 1 leader sequence; NTC9385C: from the KpnI site toa SacII site in the CMV derived exon 1 leader sequence) with the MCKenhancer, MCK promoter-exon 1 leader retaining the HTLV-I R portion ofexon 1. Purified plasmid DNA from the resultant vectors,NTC8685-MCK-muSEAP (4847 bp) and NTC9385C-MCK-muSEAP (3203 bp), wasinjected IM into one anterior tibialis muscle of 8-10 week old BALB/cfemale mice (5 mice/group), 5 μg dose in 50 μL, followed by IchorTriGrid electroporation as described in Table 1. SEAP levels in serumwas determined on day 28 (T=28) post delivery. The NTC9385C-MCK-muSEAPvector (98.4±55.8) had 4.5× higher average expression thanNTC8685-MCK-muSEAP (22.0±10.9). All 5 NTC9385C-MCK-muSEAP injected micehad higher muSEAP levels than any of the NTC8685-muSEAP mice. Thisdemonstrates that improved in vivo expression with the Nanoplasmidvectors of the invention is not specific to the CMV promoter.

Example 3: NTC9385P2, NTC9385P2a, NTC9385C2, NTC9385C2a, NTC9385R2, andNTC9385R2a Vector Construction

A series of AF eukaryotic expression vectors incorporating intronicAF-pUC origin, AF-R6K origin or AF-ColE2 replication origins aredisclosed.

FIG. 2 shows bioinformatics analysis of an intron containing the gWIZvector bacterial region (GBR2) encoded kanR selection marker-pUC origin.This intron is predicted have reduced splicing efficiency and splicingprecision due to the presence of numerous splice acceptor sites, splicedonor sites, and eukaryotic promoters in the kanR gene. Replacement ofthe kanR gene with the RNA-OUT antibiotic-free marker results in animproved intron (FIG. 3) since the RNA-OUT sequence is not predicted tocontain splice acceptor sites, splice donor sites, or eukaryoticpromoters in either orientation.

However, the pUC origin does contain an experimentally verified crypticeukaryotic promoter (FIG. 3) which likely would interfere with intronfunction. In addition, the close proximity of the pUC origin to the CMVenhancer repeats in an intronic vector is predicted to result inaberrant replication termination, resulting in replication intermediateswhich unacceptably reduce plasmid quality (Levy J. 2004. U.S. Pat. No.6,709,844). So an intronically located pUC origin would be expected tointerfere with eukaryotic intron function, and plasmid productionquality.

The R6K and ColE2 origins do not contain predicted splice acceptorsites, splice donor sites, or eukaryotic promoters in eitherorientation. Replacement of the pUC origin with the R6K or ColE2 originsresults in a improved intron design since the RNA-OUT-R6K andRNA-OUT-ColE2 bacterial region is not predicted to contain spliceacceptor sites, splice donor sites, or eukaryotic promoters in eitherorientation.

NTC9385P2 and NTC9385P2a pUC Origin Replicative Minicircle Vectors:

NTC8485-EGFP (FIG. 1) disclosed in Williams, Supra, 2010 contains theCMV enhancer and promoter upstream of a chimeric HTLV-IR rabbit β globinintron (SEQ ID NO: 1). The NTC8485-EGFP vector (FIG. 1) was linearizedwith HpaI which cuts internally within the intron (FIG. 1; SEQ ID NO: 1)leaving a blunt end. The pUC origin-RNA-OUT bacterial region was excisedfrom NTC8385 by digestion with NheI (4 bp protruding 5′ sticky end wasblunted by end filling using klenow enzyme) and KpnI (4 bp recessed 5′sticky end was blunted by end chewing using T4 DNA polymerase enzyme)(SEQ ID NO: 29). The two fragments were ligated and clones in eitherorientation (NTC8485P2-O1-EGFP or NTC8485P2-O2-EGFP) identified byrestriction mapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region and CMV enhancer encoded boundaryelement (NheI site to SpeI site; FIG. 1) was removed by digestion ofNTC8485P2-O1-EGFP and NTC8485P2-O2-EGFP with NheI and SpeI andsubsequent ligation (NheI and SpeI have compatible 4 bp sticky ends).Recombinant clones (NTC9385P2-O1-EGFP or NTC9385P2-O2-EGFP respectively)were identified by restriction mapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region (NheI site to XbaI site; FIG. 1)was removed by digestion of NTC8485P2-O1-EGFP and NTC8485P2-O2-EGFP withNheI and XbaI and subsequent ligation (NheI and XbaI have compatible 4bp sticky ends). Recombinant clones (NTC9385P2a-O1-EGFP orNTC9385P2a-O2-EGFP respectively; FIG. 4) were identified by restrictionmapping and confirmed by DNA sequencing.

The construction and isolation of these four NTC9385P clonesdemonstrates that the pUC origin and RNA-OUT selectable marker can bothfunction when located in an intron, in either orientation. Plasmidquality was evaluated by agarose gel analysis of plasmid preps from thefour intronic pUC origin-RNA-OUT vectors, and the spacer region encodedpUC-RNA-OUT vector NTC8385. Surprisingly, plasmid quality was high, andno replication intermediates were identified (FIG. 5) despite the closeproximity of the pUC origin to the CMV enhancer (Levy, Supra, 2004).

NTC9385R2 and NTC9385R2a Clones:

The NTC8485-EGFP vector (FIG. 1) was linearized with HpaI which cutsinternally within the intron (FIG. 1; SEQ ID NO: 1) leaving a blunt end.The R6K origin-RNA-OUT bacterial region was excised from NTC9385R bydigestion with NheI (4 bp protruding 5′ sticky end was blunted by endfilling using klenow enzyme) and KpnI (4 bp recessed 5′ sticky end wasblunted by end chewing using T4 DNA polymerase enzyme) (SEQ ID NO: 27).The two fragments were ligated and clones in either orientation(NTC8485R2-O1-EGFP or NTC8485R2-O2-EGFP) identified by restrictionmapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region and CMV enhancer encoded boundaryelement (NheI site to SpeI site; FIG. 1) was removed by digestion ofNTC8485R2-O1-EGFP and NTC8485R2-O2-EGFP with NheI and SpeI andsubsequent ligation (NheI and SpeI have compatible 4 bp sticky ends).Recombinant clones (NTC9385R2-O1-EGFP or NTC9385R2-O2-EGFP respectively)were identified by restriction mapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region (NheI site to XbaI site; FIG. 1)was removed by digestion of NTC8485R2-O1-EGFP and NTC8485R2-O2-EGFP withNheI and XbaI and subsequent ligation (NheI and XbaI have compatible 4bp sticky ends). Recombinant clones (NTC9385R2a-O1-EGFP orNTC9385R2a-O2-EGFP respectively; FIG. 6) were identified by restrictionmapping and confirmed by DNA sequencing.

The construction and isolation of these four NTC9385R2 derived clonesdemonstrates that the R6K origin and RNA-OUT selectable marker can bothfunction when located in an intron, in either orientation. Plasmidquality was evaluated by agarose gel analysis of plasmid preps from thefour intronic R6K origin-RNA-OUT vectors. Surprisingly, plasmid qualitywas high, and no replication intermediates were identified (not shown)despite the close proximity of the origin to the CMV enhancer (Levy,Supra, 2004).

NTC9385C2 and NTC9385C2a Clones:

The NTC8485-EGFP vector (FIG. 1) was linearized with HpaI which cutsinternally within the intron (FIG. 1; SEQ ID NO: 1) leaving a blunt end.The ColE2 origin-RNA-OUT bacterial region was excised from NTC9385C bydigestion with NheI (4 bp protruding 5′ sticky end was blunted by endfilling using klenow enzyme) and KpnI (4 bp recessed 5′ sticky end wasblunted by end chewing using T4 DNA polymerase enzyme) (SEQ ID NO: 24).The two fragments were ligated and clones in either orientation(NTC8485C2-O1-EGFP or NTC8485C2-O2-EGFP) identified by restrictionmapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region and CMV enhancer encoded boundaryelement (NheI site to SpeI site; FIG. 1) was removed by digestion ofNTC8485C2-O1-EGFP and NTC8485C2-O2-EGFP with NheI and SpeI andsubsequent ligation (NheI and SpeI have compatible 4 bp sticky ends).Recombinant clones (NTC9385C2-O1-EGFP or NTC9385C2-O2-EGFP respectively)were identified by restriction mapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region (NheI site to XbaI site; FIG. 1)was removed by digestion of NTC8485C2-O1-EGFP and NTC8485C2-O2-EGFP withNheI and XbaI and subsequent ligation (NheI and XbaI have compatible 4bp sticky ends). Recombinant clones (NTC9385C2a-O1-EGFP orNTC9385C2a-O2-EGFP respectively; FIG. 7) were identified by restrictionmapping and confirmed by DNA sequencing.

The construction and isolation of these four NTC9385C clonesdemonstrates that the ColE2 origin and RNA-OUT selectable marker canboth function when located in an intron, in either orientation. Plasmidquality was evaluated by agarose gel analysis of plasmid preps from thefour intronic ColE2 origin-RNA-OUT vectors. Surprisingly, plasmidquality was high, and no replication intermediates were identified (notshown) despite the close proximity of the origin to the CMV enhancer(Levy, Supra, 2004).

Summary:

The NTC9385P2, NTC9385P2a, NTC9385C2, NTC9385C2a, NTC9385R2, andNTC9385R2a replicative minicircle vectors are just a few possiblenonlimiting intronic bacterial region replicative minicircle vectorconfigurations. Many alternative vector configurations incorporating thenovel intronic pUC, R6K or ColE2 origin vector modifications may also bemade, including but not limited to vectors with alternative selectionmarkers, alternative promoters, alternative introns, alternativepolyadenylation sequences, a spacer region preferably less than 500 bpbetween the eukaryotic polyadenylation signal and the eukaryoticpromoter, a eukaryotic transcription terminator between the eukaryoticpolyadenylation signal and the eukaryotic promoter, S/MAR, SIDD sites,boundary elements, multiple transcription units separated by a spacerregion, and different orientations of the various vector-encodedelements or alternative R6K or ColE2 origins as described in Example 1.

An example strategy for cloning into the NTC9385R, NTC9385C, NTC9385P2,NTC9385P2a, NTC9385C2, NTC9385C2a, NTC9385R2, and NTC9385R2a etc vectorsis outlined below.

GTCGAC ATG--------Gene of interest----Stop codon------AGATCTSalI                                                 BglII

The ATG start codon (double underlined) may be immediately preceded by aunique SalI site (GTCGAC ATG). This SalI-ATG site is an effective kozaksequence for translational initiation. Alternatively, a kozaksequence-ATG (e.g. gccRccATG) may be included downstream of the SalIsite. Alternatively, the SalI site may be downstream in frame with anoptimized secretion sequence such as TPA or an alternative peptideleader such as ubiquitin, etc.

For precise cloning, genes are copied by PCR amplification from clones,cDNA, or genomic DNA using primers with SalI (5′ end) and BglII (3′ end)sites or Type IIS enzymes that create SalI or BglII compatible termini.Alternatively, genes are synthesized chemically to be compatible withthe unique SalI/BglII cloning sites in these vectors.

For all vectors one or two stop codons (preferably TAA or TGA) may beincluded after the open reading frame, prior to the BglII site.

Example 4: NTC9385P2, NTC9385P2a, NTC9385C2, NTC9385C2a, NTC9385R2, andNTC9385R2a Vector Expression

To determine intronic replicative minicircle vector eukaryotic regionfunction, transgene expression levels were determined in vitro using thevector encoded EGFP transgene. EGFP mRNA, EGFP protein (EGFPfluorescence) and mRNA splice junctions were determined after plasmidtransfection.

Adherent HEK293 (human embryonic kidney) and A549 (human lungcarcinoma),cell lines were obtained from the American Type CultureCollection (Manassas, Va., USA). Cell lines were propagated inDulbecco's modified Eagle's medium/F12 containing 10% fetal bovine serumand split (0.25% trypsin-EDTA) using Invitrogen (Carlsbad, Calif., USA)reagents and conventional methodologies. For transfections, cells wereplated on 24-well tissue culture dishes. Plasmids were transfected intocell lines using Lipofectamine 2000 following the manufacturer'sinstructions (Invitrogen, Carlsbad Calif.).

Total cellular lysates for EGFP determination were prepared byresuspending cells in cell lysis buffer (BD Biosciences Pharmingen, SanDiego, Calif., USA), lysing cells by incubating for 30 min at 37° C.,followed by a freeze-thaw cycle at −80° C. Lysed cell supernatants wereassayed for EGFP by FLX800 microplate fluorescence reader (Bio-Tek,Winooski, Vt., USA).

Cytoplasmic RNA was isolated from transfected HEK293 and A549 cellsusing the protein and RNA isolation system (PARIS kit, Ambion, AustinTex.) and quantified by A₂₆₀. Samples were DNase treated (DNA-freeDNase; Ambion, Austin Tex.) prior to reverse transcription using theAgpath-ID One step RT-PCR kit (Ambion, Austin Tex.) with the EGFPtransgene specific complementary strand primer EGFPR (FIG. 1). Intronsplicing was determined by PCR amplification of the reverse transcribedcytoplasmic RNA with the EGFP5Rseq and CMVF5seq primers (FIG. 1). EGFPmRNA levels in the reverse transcribed cytoplasmic RNA were quantifiedby quantitative PCR using a TaqMan EGFP transgene 6FAM-probe-MGBNFQprobe and flanking primers EGFPR and EGFPF (FIG. 1) in a TaqMan Geneexpression assay using Applied Biosystems (Foster City, Calif.) TaqManreagents and the Step One Real Time PCR System. Methods and primer andprobe sequences are described in Luke J M, Vincent J M, Du S X,Gerdemann U, Leen A M, Whalen R G, Hodgson C P, and Williams J A. 2011.Gene Therapy 18:334-343 included herein by reference. Linearized vectorwas used for the RT-PCR standard curve.

The results are summarized in Tables 2-6. In Table 2 EGFP expression inHEK293 and A549 cell lines after transfection with NTC8485-EGFP (spacerregion AF-pUC origin) or NTC8485 derivatives further including intronicAF-pUC, AF-ColE2 or AF-R6K origins (also with spacer region AF-pUCorigin) is shown. The ColE2 and R6K intronic bacterial regionssurprisingly had similar transgene expression levels comparable to theunaltered intron in NTC8485, while expression from the intronic pUCorigin was slightly reduced.

TABLE 2 Intron encoded RNA-OUT selection/replication origin does notprevent transgene expression NTC8485 NTC8485 A549 FU HEK293 FU VectorConstruct vector spacer Vector Intron ^(a) (T = 48 (T = 48 (all EGFP) ID# region ^(a) (intron size) mean ± SD) ^(b) mean ± SD) ^(b) NTC8485NTC-0200620 T-BH-P-AF HR-β 5886 ± 249 32628 ± 1015 (SV40-BE) (225 bpintron) (1×) (1×) NTC8485C2-O1 073-030-1H T-BH-P-AF HR ← C AF → β 3638 ±351 25231 ± 2124 (SV40-BE) (499 bp intron) (0.62×) (0.77×) NTC8485C2-O2073-030-1A T-BH-P-AF HR ← AF C → β 4144 ± 275 26233 ± 1842 (SV40-BE)(499 bp intron) (0.70×) (0.80×) NTC8485R2-O1 073-036-1B T-BH-P-AF HR ←T-R AF → β 3656 ± 240 23905 ± 679 (SV40-BE) (685 bp intron) (0.62×)(0.73×) NTC8485R2-O2 073-036-1A T-BH-P-AF HR ← AF R-T → β 4062 ± 24922165 ± 1281 (SV40-BE) (685 bp intron) (0.69×) (0.68×) NTC8485P2-O1073-041-2L T-BH-P-AF HR ← T-P-AF → β 2565 ± 294 20757 ± 1457 (SV40-BE)(1436 bp intron) (0.44×) (0.64×) NTC8485P2-O2 073-041-2E T-BH-P-AF HR ←AF P-T → β 2411 ± 320 15333 ± 1145 (SV40-BE) (1436 bp intron) (0.41×)(0.47×) ^(a) trpA term = T; HTLV-IR = HR; B globin 3′ acceptor site = β;RNA-OUT selectable marker = AF; PAS-BH = BH; pUC origin = P; R6K origin= R; ColE2 origin = C; CMV boundary element (XbaI-SpeI fragment) = BE;SV40 enhancer = SV40. Bracketed BE and or SV40 are spacer regionflanking eukaryotic sequences ^(b) Fluorescence units = FU ( ) Mean FUstandardized to NTC8485

Conversion of the NTC8485C2, NTC8485R2 and NTC8485P2 (pUC origin-AFspacer region) vectors into replicative minicircles by removal of thepUC origin-AF spacer region to create the corresponding NTC9385C2,NTC9385R2 and NTC9385P2 vectors (Example 3) unexpectedly dramaticallyincreased expression compared to the NTC8485C2, NTC8485R2 and NTC8485P2parent vectors (Table 3). This demonstrates that the replicativeminicircles of the invention unexpectedly improve expression throughremoval of vector spacer region encoded bacterial region.

TABLE 3 Improved expression with intron encoded RNA-OUTselection/replication origin A549 FU ^(b) HEK293 FU ^(b) VectorConstruct Vector Spacer (T = 48 (T = 48 (all EGFP) ID # Region ^(a)Vector Intron ^(a) mean ± SD) mean ± SD) NTC8485 NTC- T-BH-P-AF HR-β5886 ± 249 32628 ± 1015 0200620 (SV40-BE) (1×) (1×) NTC9385C 071-020-2DC-AF HR-β 8591 ± 168 35293 ± 1798 (1.46×) (1.08×) NTC8485C2-O1073-030-1H T-BH-P-AF HR ← C AF → β 3638 ± 351 25231 ± 2124 (SV40-BE)(0.62×) (0.77×) NTC8485C2-O2 073-030-1A T-BH-P-AF HR ← AF C → β 4144 ±275 26233 ± 1842 (SV40-BE) (0.70×) (0.80×) NTC9385C2-O1 073-032-5A NoneHR ← C AF → β 6793 ± 521 24762 ± 1498 (1.15x) (0.76×) NTC9385C2-O2073-032-6A None HR ← AF C → β 7330 ± 692 24811 ± 1256 (1.25×) (0.76×)NTC9385C2a-O1 073-032-7A None (BE) HR ← C AF → β 7515 ± 282 29444 ± 2193(1.28×) (0.90×) NTC9385C2a-O2 073-032-8A None (BE) HR ← AF C → β 7255 ±322 27055 ± 1850 (1.23×) (0.83×) NTC9385R 071-025-2C T-R-AF HR-β 5813 ±949 29822 ± 661 (0.99×) (0.91×) NTC8485R2-O1 073-036-1B T-BH-P-AF HR ←T-R AF → β 3656 ± 240 23905 ± 679 (SV40-BE) (0.62×) (0.73×) NTC8485R2-O2073-036-1A T-BH-P-AF HR ← AF R-T → β 4062 ± 249 22165 ± 1281 (SV40-BE)(0.69×) (0.68×) NTC9385R2-O1 073-038-1A None HR ← T-R-AF → β 10959 ±1278 34521 ± 3694 (1.86×) (1.06×) NTC9385R2-O2 073-038-2A None HR ← AFR-T → β 10652 ± 567 31586 ± 1121 (1.81×) (0.97×) NTC9385R2a-O1073-038-3A None (BE) HR ← T-R-AF → β 10699 ± 674 37603 ± 2671 (1.82×)(1.15×) NTC9385R2a-O2 073-038-4A None (BE) HR ← AF R-T → β 10251 ± 134334086 ± 1518 (1.74×) (1.04×) NTC8485P2-O1 073-041-2L T-BH-P-AF HR ←T-P-AF → β 2565 ± 294 20757 ± 1457 (SV40-BE) (0.44×) (0.64×)NTC8485P2-O2 073-041-2E T-BH-P-AF HR ← AF P-T → β 2411 ± 320 15333 ±1145 (SV40-BE) (0.41×) (0.47×) NTC9385P2-O2 073-043-1A None HR ← AF P-T→ β 5561 ± 497 21838 ± 589 (0.94×) (0.67×) NTC9385P2a-O2 073-043-2A None(BE) HR ← AF P-T → β 6291 ± 544 23808 ± 2411 (1.07×) (0.73×) ^(a) trpAterm = T; HTLV-IR = HR; B globin 3′ acceptor site = β; RNA-OUTselectable marker = AF; PAS-BH = BH; pUC origin = P; R6K origin = R;ColE2 origin = C; CMV boundary element (XbaI-SpeI fragment) = BE; SV40enhancer = SV40. Bracketed BE and or SV40 are spacer region flankingeukaryotic sequences ^(b) Fluorescence units = FU ( ) Mean FUstandardized to NTC8485

Table 4 demonstrates that mRNA splicing is accurate and spliced mRNAexport efficient with the intronic bacterial regions encoded inNTC9385C2, NTC9385R2 and NTC9385P2. A minor amount of a cryptic 209 bppUC origin derived exon was identified with NTC9385P2-O2 (but notNTC9385P2-O1) in A549 cells but not HEK293 cells (Table 4; 490 bp band).The cryptic exon sequence was determined by sequencing of the PCRproduct and the cryptic 209 bp exon utilized cryptic splice donor andacceptor sites within the pUC origin (FIG. 3).

TABLE 4 Intron functional analysis - Splicing accuracy and exportefficiency EGFP % Predicted Actual mRNA EGFP spliced exon spliced exon #Plasmid Cell line RT-PCR (pg) mRNA ^(c) size (unspliced) size (PCR) 1NTC8685 HEK293 448.3 ± 38.7  0.74% 279 (514) 279 ^(a) 2 NTC9385C2-O1HEK293 274.6 ± 10.3  0.44% 279 (788) 279 ^(a) 3 NTC9385C2-O2 HEK293227.4 ± 4.9   0.41% 279 (788) 279 ^(a) 4 NTC9385R2-O1 HEK293 398.9 ±14.8  0.64% 279 (974) 279 ^(a) 5 NTC9385R2-O2 HEK293 350.7 ± 5.2   0.57%279 (974) 279 ^(a) 6 NTC9385P2-O2 HEK293 181.6 ± 5.5   0.30% 279 (974)279 ^(a) 7 NTC8485P2-O1 HEK293 160.1 ± 12.3  0.27%  279 (1715) 279 ^(a)8 NTC8685 A549 50.6 ± 4.3 0.128% 279 (514) 279 ^(a) 9 NTC9385C2-O1 A54929.2 ± 1.2 0.082% 279 (788) 279 10 NTC9385C2-O2 A549 23.9 ± 1.4 0.074%279 (788) 279 11 NTC9385R2-O1 A549 41.9 ± 1.6 0.116% 279 (974) 279 12NTC9385R2-O2 A549 35.8 ± 2.2 0.096% 279 (974) 279 ^(a) 13 NTC9385P2-O2A549 17.4 ± 0.6 0.050%  279 (1715) 279 ^(b) 14 NTC8485P2-O1 A549  7.0 ±0.2 0.024%  279 (1715) 279 ^(a) Correct splice junction verified by DNAsequencing of PCR product ^(b) Faint extra bands at 490 and 650 bp, notpresent in 6 (HEK293 equivalent) or other samples ^(c) % of totalcytoplasmic RNA that is EGFP mRNA

Table 5 further demonstrates unexpectedly robust expression is observedwith all NTC9385C2, NTC9385R2 and NTC9385P2 replicative minicirclevectors (both orientations, with and without CMV boundary region).Overall, the highest expression is obtained with the R6K replicativeminicircle vectors (NTC9385R2-O1; NTC9385R2-O2; NTC9385R2a-O1;NTC9385R2a-O2).

TABLE 5 Intron vector expression efficiency Vector A549 FU HEK293 FUVector Construct Spacer (T = 48 (T = 48 # (all EGFP) ID # Region ^(a)Vector Intron ^(a) mean ± SD) ^(b) mean ± SD) ^(b) 1 NTC9385C2-O1073-032-5A None HR ← C AF → β  7581 ± 1145 20868 ± 9153 2 NTC9385C2-O2073-032-6A None HR ← AF C → β 6012 ± 503 12902 ± 2356 3 NTC9385C2a-O1073-032-7A (BE) HR ← C AF → β 6018 ± 979 13564 ± 799  4 NTC9385C2a-O2073-032-8A (BE) HR ← AF C → β 6633 ± 136 16119 ± 729  5 NTC9385R2-O1073-038-1A None HR ← T-R AF → β 9626 ± 304 18627 ± 999  6 NTC9385R2-O2073-038-2A None HR ← AF R-T → β 8513 ± 235 12660 ± 348  7 NTC9385R2a-O1073-038-3A (BE) HR ← T-R AF → β 8295 ± 188 15601 ± 2550 8 NTC9385R2a-O2073-038-4A (BE) HR ← AF R-T → β 8724 ± 188 19219 ± 1763 9 NTC9385P2-O1073-126-1A None HR ← T-P-AF → β 6086 ± 704 16967 ± 2237 10 NTC9385P2-O2073-043-1A None HR ← AF P-T → β 4941 ± 283 11604 ± 2580 11 NTC9385P2a-O1073-126-2A (BE) HR ← T-P-AF → β 5277 ± 114 13073 ± 1779 12 NTC9385P2a-O2073-043-2A (BE) HR ← AF P-T → β 5122 ± 608 11182 ± 870  ^(a) trpA term =T; HTLV-IR = HR; B globin 3′ acceptor site = β; RNA-OUT selectablemarker = AF; pUC origin = P; R6K origin = R; ColE2 origin = C; CMVboundary element (XbaI-SpeI fragment) = BE. Bracketed BE is spacerregion flanking eukaryotic sequences ^(b) Fluorescence units = FU

Table 6 demonstrates in vivo expression after intradermal delivery withthe intronic bacterial region vectors of the invention was improvedcompared to an optimized plasmid comparator (NTC8685). NTC9385R2a-O2expression was surprisingly improved 1.9-4.1 fold compared to NTC8685while NTC9385P2a-O1 was unexpectedly improved 5.0-8.0 fold. NTC9385R2-O2expression was also improved (1.3-1.5 fold compared to NTC8685) but lessthan NTC9385R2a-O2 suggesting that the CMV promoter derived boundaryelement adjacent to the spacer region is beneficial. Replicativeminicircle expression is surprisingly much higher relative to plasmidcomparator in vivo compared to in vitro (Table 6). While not limitingthe application of the invention, this may be an unexpected benefit ofremoval of the large spacer region encoded replication origin andselectable marker, perhaps through rapid spacer region directedheterochromatin formation that is more prevalent in vivo than in vitro.

The improved transgene expression level after intradermal deliverydemonstrates the application of Nanoplasmid and replicative minicirclevectors of the invention for cutaneous DNA vaccination and gene therapyapplications. For example, for intradermal DNA vaccination, epidermalDNA vaccination, or transcutaneous DNA vaccination using a variety ofantigens. For example, for gene therapy applications such as woundhealing, burns, diabetic foot ulcer, critical limb ischemia therapies,or cosmetic treatment for different dermatological conditions, includinganti-aging (anti-wrinkle), scar revision, radiation induced lesions,hair growth, surgical skin graft enhancement, or hemangioma, usinggrowth factors such as hypoxia inducible factor, hypoxia induciblefactor 1α, keratinocyte growth factor, vascular endothelial growthfactor (VEGF), fibroblast growth factor-1 (FGF-1, or acidic FGF), FGF-2(also known as basic FGF), FGF-4, placental growth factor (P1GF),angiotensin-1 (Ang-1), hepatic growth factor (HGF), DevelopmentallyRegulated Endothelial Locus (Del-1), stromal cell derived factor-1(SDF-1), etc.

TABLE 6 Replicative minicircle vector expression in vitro(lipofectamine) and in vivo (intradermal delivery with electroporation)ID + EP ^(c) ID + EP ^(c) ID + EP ^(c) (pg/mL) (pg/mL) (pg/mL) muSEAP SRA549 HEK-293 T = 4 day T = 7 day T = 14 day Vector ^(b) SR ^(a) (bp)Intron ^(a) (A₄₀₅) ^(d) (A₄₀₅) ^(d) mean ± SD mean ± SD mean ± SDNTC8685 T-VA1- 1695 HR-β 0.240 ± 0.029 3.002 ± 0.188 1.9 ± 1.2 6.7 ± 4.15.0 ± 3.9 BH-P-AF (1.0×) (1.0×) (1.0×) (1.0×) (1.0×) (SV40) NTC9385 None0 HR T ← P 0.467 ± 0.047 2.890 ± 0.085 9.5 ± 6.2 53.4 ± 51.5 34.8 ± 29.6P2a-O1 (BE) AF → -β (2.0×) (1.0×) (5.0×) (8.0×) (7.0×) NTC9385 None 0 HR← AF 0.409 ± 0.039 2.561 ± 0.038 6.5 ± 6.1 27.6 ± 25.9  9.5 ± 11.2R2a-O2 (BE) R → T-β (1.7×) (0.9×) (3.4×) (4.1×) (1.9×) NTC9385 None 0 HR← AF 0.564 ± 0.008 2.999 ± 0.106 2.8 ± 3.8  8.8 ± 15.7 7.0 ± 8.5 R2-O2 R→ T-β (2.4×) (1.0×) (1.5×) (1.3×) (1.4×) ^(a) Prokaryotic terminator =T; HTLV-IR = HR; B globin 3′ acceptor site = β; RNA-OUT = AF; pUC origin= P; R6Kγ origin = R; ColE2-P9 origin = C; CMV boundary element(XbaI-SpeI fragment) = BE; SV40 enhancer = SV40; PAS-BH = BH. BracketedBE or SV40 are spacer region flanking eukaryotic sequences ^(b) Pvectors produced in dcm- XL1B1ue NTC54208; R vectors produced in dcm-R6K Rep cell line NTC711231; C vectors produced in dcm- ColE2 Rep cellline NTC710351 ^(c) Dose = 50 μg in 50 μL saline injected intradermally(ID) with EP. 6 mice/group. Mean ± SD pg/mL muSEAP on indicated day postEP reported. ( ) Mean muSEAP standardized to NTC8685 ^(d) muSEAP plasmidDNA transfected with Lipofectamine 2000. Mean ± SD 48 hr posttransfection A₄₀₅ reported. ( ) Mean A₄₀₅ standardized to NTC8685

Reduction of the vector spacer region size as described herein byremoval of the bacterial region replication origin and addition of anintronic R6K, ColE2, pUC or P_(min) pUC origin vectors of the inventionwill also increase the duration of in vivo expression since expressionduration is improved with plasmid vectors in which the bacterial regionis removed (minicircle) or replaced with a spacer region of up to atleast 500 bp (Lu et al., Supra, 2012). Thus the replicative minicirclevectors of the invention also have additional utility for applicationsrequiring extended duration expression, such as: liver gene therapyusing hydrodynamic delivery with transgenes such as α-1 antitrypsin(AAT) for AAT deficiency, Coagulation Factor VIII for Hemophilia ATherapy or Coagulation Factor IX for Hemophilia B Therapy etc: lung genetherapy with transgenes such as Cystic fibrosis transmembraneconductance regulator (CFTR) for cystic fibrosis etc; muscle genetherapy with transgenes such as the GNE gene for Hereditary inclusionbody myopathies (HIBM), or dystrophin or dystrophin minigenes forduchenne muscular dystrophy (DMD), etc.

The intronic replicative minicircles of the invention spacer regionbetween the 5′ and 3′ ends of the eukaryotic region may optionallyencode up to 500 bp of sequence. This spacer region may include a numberof functional sequences such as bacterial or eukaryotic selectablemarkers, bacterial or eukaryotic replication origins, bacterialtranscription terminators, eukaryotic transcription terminators,supercoiling-induced DNA duplex destabilized (SIDD) structures, boundaryelements, S/MARs, RNA Pol I or RNA Pol III expressed sequences or otherfunctionalities.

Example 5: Replicative Minicircle Vector Fermentation Production

Fermentation:

Fermentations were performed using proprietary fed-batch media (NTC3019,HyperGRO media) in New Brunswick BioFlo 110 bioreactors as described(Carnes and Williams, Supra, 2011). The seed cultures were started fromglycerol stocks or colonies and streaked onto LB medium agar platescontaining 6% sucrose. The plates were grown at 30-32° C.; cells wereresuspended in media, and used to provide approximately 0.1% inoculumsfor the fermentations that contained 0.5% sucrose to select for RNA-OUTplasmids.

Production Hosts:

Antibiotic-free pUC origin RNA-OUT plasmid fermentations were performedin E. coli strain XL1Blue [recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1lac [F′ proAB lacIqZΔM15 Tn10 (Tet^(r))] (Stratagene, La Jolla, Calif.)]dcm or DH5α [F-Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK−,mK+) phoA supE44 λ-thi-1 gyrA96 relA1] (Invitrogen, Carlsbad Calif.) dcmcontaining chromosomally integrated pCAH63-CAT RNA-IN-SacB (P5/6 6/6) asdisclosed in Williams, Supra, 2008. SacB (Bacillus subtilislevansucrase) is a counterselectable marker which is lethal to E. colicells in the presence of sucrose. Translation of SacB from theRNA-IN-SacB transcript is inhibited by plasmid encoded RNA-OUT. Thisfacilitates plasmid selection in the presence of sucrose, by inhibitionof SacB mediated lethality. These production strains areNTC54208=XL1Blue dcm attλ::P5/6 6/6-RNA-IN-SacB and NTC48165=DH5α dcmattλ::P5/6 6/6-RNA-IN-SacB

Antibiotic-free R6K plasmid propagation and fermentations were performedusing pL promoter heat inducible ‘P42L, P106L and F107S’ or ‘P42L,P113S’ π copy number mutant cell line lines, the creation of which isdisclosed in patent application PCT/US 13/00068 (Filing No. 61/743,219)entitled ‘DNA plasmids with improved expression’ and included herein byreference. NTC711231 is NTC54208-pR pL (OL1-G to T) P42L-P106L-F107S(P3-). NTC54208=XL1Blue dcm attλ::P5/6 6/6-RNA-IN-SacB.NTC731871=NTC54208-pR pL (OL1-G to T) P42L-P113S (P3-).NTC711772=NTC48165-pR pL (OL1-G to T) P42L-P106L-F107S (P3-).NTC48165=DH5α dcm attλ::P5/6 6/6-RNA-IN-SacB.

Antibiotic-free ColE2 plasmid propagation and fermentations wereperformed using pL promoter heat inducible ‘G194D’ Rep protein copynumber mutant cell line NTC710351 the creation of which is disclosed inpatent application PCT/US 13/00068 (Filing No. 61/743,219) entitled ‘DNAplasmids with improved expression’ and included herein by reference.NTC710351=NTC54208-pR pL (OL1-G to T) ColE2 Rep G194D

Analytical Methods:

Culture samples were taken at key points and at regular intervals duringall fermentations. Samples were analyzed immediately for biomass (OD₆₀₀)and for plasmid yield. Plasmid yield was determined by quantification ofplasmid obtained from Qiagen Spin Miniprep Kit preparations as described(Carnes and Williams, Supra, 2011). Briefly, cells were alkaline lysed,clarified, plasmid was column purified, and eluted prior toquantification. Agarose gel electrophoresis analysis (AGE) was performedon 0.8-1% Tris/acetate/EDTA (TAE) gels as described in Carnes andWilliams, Supra, 2011.

Results:

Fermentation yields are summarized in Table 7. The results demonstratedthat the replicative minicircle vectors of the invention have efficientmanufacture. Manufacture was effective with ColE2, R6K and pUCreplicative minicircles with yield of 184-745 mg/L, up to >100 foldimproved compared to reported yields of 5 mg/L with alternative shortspacer region minicircle vectors (Kay et al., Supra, 2010).Additionally, the replicative minicircle vectors of the invention do notrequire the complicated difficult to scale expensive additionalmanufacturing steps required to remove the large bacterial regionbetween the eukaryotic polyA and promoter with minicircle vectors (Kayet al., Supra, 2010) since replicative minicircles have less than 500 bpbetween the eukaryotic polyA and the promoter which does not need to beremoved.

TABLE 7 Intronic RNA-OUT AF selection plasmid fermentation yields ^(a)Growth phase Production Specific phase final Production ProductionPlasmid Origin/ yield Specific phase final phase final size host Vector(mg/L/ yield (mg/ biomass yield Plasmid (kb) strain type ^(f) OD600)L/OD₆₀₀) (OD₆₀₀) (mg/L) ^(e) NTC9385R-EGFP 2.4 R6K ^(b) SR 1.4-1.5 4.489 392 NTC9385R-L-CF 3.4 R6K ^(b) SR 2.2-2.4 8.5 72 615 NTC9385R-L-CF3.4 DR6K ^(b) SR 2.3-2.6 7.8 98 745 NTC9385R2-O1-EGFP 2.4 R6K ^(b)Intron 1.4-1.5 6.3 66 414 NTC9385R2-O2-EGFP 2.4 R6K ^(b) Intron 1.1-1.83.8 70 269 NTC9385R2-O2-EGFP 2.4 DR6K ^(b) Intron 1.4 4.1 93 376NTC9385R2a-O1-EGFP 2.5 R6K ^(b) Intron 1.7-1.9 4.8 74 356NTC9385R2a-O2-EGFP 2.5 R6K ^(b) Intron 1.0-1.8 3.5 69 244NTC9385R2b-O2-EGFP 2.4 R6K ^(b) Intron 2.6-3.0 7.2 61 440 (AF-SR)NTC9385Ra-O1-EGFP 2.4 R6K ^(b) SR 2.8-3.4 7.0 49 340 (AF-intron)NTC9385Ra-O1-EGFP 2.4 DR6K ^(b) SR 1.9-2.2 6.3 99 623 (AF-intron)NTC9385Ra-O2-EGFP 2.4 R6K ^(b) SR 1.2-1.7 4.6 40 184 (AF-intron)NTC9385RbF-EGFP 2.5 R6K ^(b) 3′ UTR 2.9 4.5 56 258 (AF-intron)NTC9385RbF-EGFP 2.5 DR6K ^(b) 3′ UTR 2.0-2.4 4.2 99 420 (AF-intron)NTC9385C-EGFP 2.2 ColE2 ^(c) SR 0.6-1.0 5.8 115 672 NTC9385C2a-O1-EGFP2.3 ColE2 ^(c) Intron 0.8-1.1 3.7 87 323 NTC9385C2a-O2-EGFP 2.3 ColE2^(c) Intron 0.8-1.0 3.7 64 235 NTC9385P2a-O1-EGFP 3.2 pUC ^(d) Intron1.2-1.3 8.6 82 703 NTC9385P2a(0.85)-O1-EGFP 2.9 P_(min) ^(d) Intron0.9-1.0 5.6 84 468 NTC9385P2a(0.85)-O2-EGFP 2.9 P_(min) ^(d) Intron 0.8-1.05 5.7 77 439 NTC8385(0.85)-EGFP 2.8 P_(min) ^(d) SR 0.9 6.4 105667 ^(a) 30° C. growth phase to 50-60 OD600. Plasmid copy number theninduced by temperature shift to 42° C. and subsequent 7-10.5 hr growthpost induction (production phase) ^(b) R6K plasmid produced in: R6K =cell line NTC711231 = NTC54208-pR pL (OL1-G to T) P42L-P106L-F107S(P3-). NTC54208 = XL1Blue dcm attλ::P5/6 6/6-RNA-IN- SacB. DR6K =NTC711772 = NTC48165-pR pL (OL1-G to T) P42L-P106L-F107S (P3-). NTC48165= DH5α dcm attλ::P5/6 6/6-RNA-IN- SacB. ^(c) ColE2 plasmid produced incell line NTC710351 = NTC54208-pR pL (OL1-G to T) ColE2 Rep G194D ^(d)pUC and P_(min) plasmid produced in cell line NTC54208 = XL1Blue dcmattλ::P5/6 6/6-RNA-IN- SacB ^(e) By comparison, minicircle manufacturingfinal volumetric yield are 5 mg/L (Kay et al., Supra, 2010) f SR =Replication origin and selectable marker in spacer region. Intron =Replication origin and selectable marker in intron. NTC9385R2b hasRNA-OUT in SR and R6K origin in intron. NTC9385Ra has RNA-OUT in SR andR6K origin in SR. NTC9385RbF has R6K in 3′ UTR and RNA-OUT in SR.

As well, fermentation plasmid DNA was high quality with ColE2, R6K andpUC replicative minicircles. A comparison of plasmid production with the4 R6K intronic selection vectors (NTC9385R2-O1-EGFP; NTC9385R2-O2-EGFP;NTC9385R2a-O1-EGFP; NTC9385R2a-O2-EGFP) versus a standard R6K spacerregion vector NTC9385R-EGFP demonstrated no differences in yield (Table7) or quality (FIG. 8).

Example 6: High Level Expression with Replicative Minicircle VectorsModified to Include an RNA Selectable Marker or a EukaryoticTranscriptional Terminator in the Spacer Region

Minicircle vectors contain a spacer region between the eukaryotic regionpolyA and promoter sequences; this spacer region may be at least 500 bp(Lu et al., Supra, 2012). To determine if the spacer region may encode aselectable marker, bacterial transcription terminator, eukaryotictranscription terminator or other functionality, replicative minicirclevectors were created that included either a RNA-OUT selectable marker ora Gastrin eukaryotic terminator. FIG. 9 shows NTC9385R2b-O2-EGFP, anexample intronic R6K origin replicative minicircle vector in which theRNA-OUT RNA selectable marker is located in the spacer region betweenthe eukaryotic polyA and the eukaryotic promoter rather than within theintron. The vector has a 148 bp spacer region, well below 500 bp. Thevector was transfected into HEK293 and A549 cell lines, and EGFPexpression and splicing were analyzed as described in Example 4. Highlevel expression (Table 8) and accurate splicing (Table 9) were observedwith this vector. This vector could be further modified to replaceRNA-OUT with a different RNA selectable marker, such as pMB1 RNAI, ColE2RNAI, IncB RNAI, RSM, etc.

Improved transgene expression was observed when the gastrin eukaryotictranscription terminator was inserted into the spacer region(NTC9385R2a-O2-Gt versus NTC9385R2a-O2; Table 8). Collectively, theseresults demonstrate additional functionalities may be added to thespacer region without interfering with replicative minicircleperformance

Additional sequences that may be added to the spacer include bacterialselectable markers (e.g. RNA-OUT or RNAI or alternative RNA selectablemarkers; see Examples 7 and 9), eukaryotic selectable markers, bacterialtranscription terminators, eukaryotic transcription terminators (e.g.gastrin terminator), boundary elements, supercoiling-induced DNA duplexdestabilized (SIDD) structures, S/MARs, RNA Pol I or RNA Pol IIIexpressed sequences or other functionalities. As well, additionalsequences could be encoded within the intron, such as SIDD structures,RNA Pol III transcription units expressing short hairpin RNA's orimmunostimulatory RNAs such as those disclosed in Williams, Supra, 2008,included herein by reference.

Example 7: RNAI Regulated Vectors

Alternative RNA selectable markers known in the art may be utilized inreplicative minicircle vectors. For example, RNA-OUT (RNA-IN regulatedchromosomal selection marker) may be replaced with the pMB1 plasmidorigin encoded RNAI (RNAII regulated chromosomal selection markerGrabherr and, Pfaffenzeller Supra, 2006; Cranenburgh, Supra, 2009),plasmid pMU720 origin encoded RNAI (SEQ ID NO: 35) that represses RNA IIregulated targets (Wilson et al., Supra, 1997), plasmid R1ParB locus Sok(Hok regulated chromosomal selection marker; Morsey, Supra, 1999), Fplasmid Flm locus FlmB (flmA regulated chromosomal selection marker;Morsey, Supra, 1999) or other RNA selectable markers described in theart. The use of alternative RNA selectable markers to constructreplicative minicircles was demonstrated here by substitution of RNA-OUTwith the pMB1 plasmid origin encoded RNAI and assessing expression andsplicing accuracy.

RNAI is present within the intron of the NTC9385P2 and NTC9385P2avectors (FIG. 3; FIG. 4) and NTC9385P2(0.85) and NTC9385P2a(0.85)vectors (Example 8). The observed accurate splicing (Table 4) and robustexpression (Table 5) of NTC9385P2 clones with RNAI in either orientationdemonstrated that intronic pMB1 plasmid origin encoded RNAI expressionis compatible with replicative minicircle function. The increased invivo expression observed with NTC9385P2a-O1-muSEAP (Table 6) furtherdemonstrates that intronic pMB1 plasmid origin encoded RNAI expressionis compatible with replicative minicircle function. The observedaccurate splicing (Table 9) and robust expression (Table 11) ofNTC9385P2(0.85) clones with RNAI in either orientation demonstrated thatintronic pMB1 plasmid origin encoded RNAI expression is compatible withreplicative minicircle function (see Example 10).

Nanoplasmid variants with the pMB1 antisense RNA RNAI (SEQ ID NO:33)with promoter and terminator region (RNAI selectable marker: SEQ IDNO:34 flanked by DraIII-KpnI restriction sites for cloning as describedpreviously for RNA-OUT) substituted for RNA-OUT were constructed asdescribed in Example 3 and tested for expression to determine ifalternative selectable markers may be utilized in place of RNA-OUT. Theresults (Table 8) demonstrate alternative RNA selectable markers may besubstituted for RNA-OUT. Substitution of RNAI for RNA-OUT in the vectorspacer region (NTC9385Ra-RNAI-O1) or in the intron in either orientation(NTC9385R-RNAI-O1 and NTC9385R-RNAI-O2) did not reduce expressionrelative to the corresponding RNA-OUT construct. To determine splicingaccuracy, NTC9385R-RNAI-O1-EGFP and NTC9385R-RNAI-O2-EGFP weretransfected into the A549 cell line and cytoplasmic RNA isolated fromtransfected A549 cells using the protein and RNA isolation system (PARISkit, Ambion, Austin Tex.) and quantified by A₂₆₀. Samples were DNasetreated (DNA-free DNase; Ambion, Austin Tex.) prior to reversetranscription using the Agpath-ID One step RT-PCR kit (Ambion, AustinTex.) with the EGFP transgene specific complementary strand primer EGFPR(FIG. 1). Intron splicing was determined by PCR amplification of thereverse transcribed cytoplasmic RNA with the EGFP5Rseq and CMVF5seqprimers (FIG. 1). The resultant PCR product (a single band in each case)was determined by sequencing to be the correct spliced exon1-exon2fragment (Table 9). This demonstrated that, like intronic RNA-OUT,intronic RNAI in either orientation is accurately removed by splicingand does not interfere with splicing accuracy. This further demonstratesthat alternative RNA based selectable markers may be substituted forRNA-OUT in the spacer region or the intron and that pMB1 RNAI is apreferred RNA based selectable marker for replicative minicirclevectors.

Alternatively, the 108 bp RNAI antisense repressor RNA (SEQ ID NO: 33)may be substituted for the 70 bp RNA-OUT antisense repressor RNA (SEQ IDNO: 21) retaining the flanking RNA-OUT transcription control sequencesin any of the constructs described in Examples 2-11. RNAI regulatedreplicative minicircle vectors may be grown in RNAII-SacB regulated celllines further expressing, as required, R6K, ColE2-P9, or ColE2 relatedRep protein. RNAII-SacB regulated cell lines may be made replacing theRNA-IN sequence in pCAH63-CAT RNA-IN-SacB (P5/6 6/6) with a RNAII targetsequence as described in Williams, Supra, 2008 included herein byreference. Alternatively, RNAI regulated replicative minicircle vectorsmay be grown in any of the RNAII regulated chromosomal selection markercell lines disclosed in Grabherr and Pfaffenzeller, Supra, 2006 andCranenburgh, Supra, 2009. These cell lines would be modified forexpression, as required, of R6K, ColE2-P9, or ColE2 related Rep protein.

TABLE 8 High level expression with vectors with pMB1 RNAI encoded in thespacer region or intron A549 FU^(c) HEK293 FU^(c) Vector SR ^(d) (T = 48(T = 48 (all EGFP) Spacer region ^(a) (bp) Intron ^(a) mean + SD) mean +SD) NTC8685 T-VA1 1465 HR-β ^(b) 8546 ± 1163 62068 ± 1760 BH-P-AF(1.00×) (1.00×) (SV40) NTC8385 T-P_(min)-AF 866 HR-β ^(b) 9364 ± 96631482 ± 1822 (0.85 kb) ^(e) (1.10×) (0.51×) NTC9385C ← C-AF → 281 HR-β^(b) 8860 ± 382 33356 ± 1489 (1.04×) (0.54×) NTC9385R ← T-R-AF → 466HR-β ^(b) 16237 ± 2520 55919 ± 6371 (1.90×) (0.90×) NTC9385Ra-O2 ← T-R306 HR- ← AF-β 14510 ± 835 49526 ± 2179 (1.70×) (0.80×) NTC9385R2-O2None 0 HR ← AF R-T → -β 15394 ± 683 30995 ± 4487 (1.80×) (0.50×)NTC9385R2a-O2 (BE) 0 HR ← AF R-T → -β 11383 ± 253 36382 ± 1086 (1.33×)(0.59×) NTC9385R2a-O2-Gt TT-(BE) 73 HR ← AF R-T → -β 15076 ± 321 49289 ±2672 (1.76×) (0.79×) NTC9385R2b-O2 AF→ 148 HR ← R-T → -β 10721 ± 103942507 ± 5321 (1.25×) (0.68×) NTC9385Ra-O1 ← T-R-AF → 466 HR-AF → -β13929 ± 1291 56552 ± 2714 dual (1.63×) (0.91×) NTC9385Ra-O2 ← T-R-AF →466 HR- ← AF-β 12543 ± 245 54379 ± 1244 dual (1.47×) (0.89×) NTC9385Ra-← T-R-RNAI → 488 HR-AF → -β 15773 ± 238 55468 ± 6619 RNAI-O1 (1.85×)(0.89×) NTC9385R- ← T-R-AF → 466 HR- ← RNAI-β 14296 ± 287 60630 ± 2176RNAI-O1 (1.67×) (0.98×) NTC9385R- ← T-R-AF → 466 HR-RNAI → -β 12271 ±466 60691 ± 6482 RNAI-O2 (1.44×) (0.98×) ^(a) trpA term = T; Gastrin(Gt) eukaryotic terminator = TT; HTLV-IR = HR; B globin 3′ acceptor site= β; RNA-OUT selectable marker = AF; pUC origin RNAI antisense RNAselectable marker = RNAI; pUC origin = P; R6K origin = R; ColE2 origin =C; CMV boundary element = BE; PAS-BH = BH; SV40 enhancer = SV40.Bracketed BE or SV40 are spacer region flanking eukaryotic sequences^(b) HR β intron is 225 bp ^(c)EGFP plasmid DNA transfected withLipofectamine 2000. Mean ± SD Fluorescence units (FU) at 48 hrs posttransfection reported. ( ) Mean FU standardized to NTC8685 ^(d) SpacerRegion (SR) size (bp) is total bp of components between polyA and CMV orSV40 enhancer, and does not include the SV40 enhancer or BE. ^(e)P_(min) minimal pUC origin (SEQ ID NO: 45) and RNA-OUT (bacterial region= SEQ ID NO: 46)

TABLE 9 Accurate splicing with replicative minicircle vectors with pMB1RNAI and minimal pUC origin encoded in the intron EGFP % Predictedspliced Actual spliced Cell RT-PCR EGFP exon size exon size Plasmid line(pg) mRNA (unspliced) (PCR) NTC9385R A549 87.5 ± 3.8 0.260% 279 (514)279 ^(c) NTC9385R- A549 45.9 ± 3.0 0.142% 279 (667) 279 ^(c) RNAI-O1NTC9385R- A549 43.1 ± 2.1 0.121% 279 (667) 279 ^(c) RNAI-O2 NTC9385R2-O2A549 26.8 ± 2.2 0.094% 279 (974) 279 ^(c) NTC9385Ra-O2 A549 60.2 ± 6.10.198% 279 (648) 279 ^(c) NTC9385R2b-O2 A549 42.1 ± 1.2 0.148% 279 (819)279 ^(c) NTC9385P2a-O1 A549 15.8 ± 1.3 0.054% 279 (1715) 279 ^(c)NTC9385P2a-O2 A549 11.6 ± 0.2 0.037% 279 (1715) 279 ^(a) NTC9385P2a A54920.1 ± 1.6 0.085% 279 (1366) 279 ^(c) (0.85)-O1 NTC9385P2a A549  6.7 ±0.5 0.027% 279 (1366) 279 ^(b) (0.85)-O2 NTC9385C- A549 0.003 0.000 Noband No band C2x4-muSEAP (negative control) ^(a) Faint extra bands at490 and 650 bp (previously observed with transfection of NTC9385P2a-O2into A549; (see Table 4). Correct splice junction verified by DNAsequencing of PCR product. Faint band at 490 bp corresponds to mRNA withan additional pUC derived exon (see FIG. 3) ^(b) Very faint extra bandsat 490 and 650 bp. Correct splice junction verified by DNA sequencing ofPCR product. Faint band at 490 corresponds to mRNA with an additionalpUC derived exon (see FIG. 3) ^(c) Correct splice junction verified byDNA sequencing of PCR product

Example 8: Spacer Region and Intron Modified Nanoplasmid Vectors

NTC8685 (SR=1465 bp) has much lower in vivo expression than NTC9385R(SR=466 bp) and NTC9385C (SR=281 bp) (Table 1). A minimal pUC originvector was constructed with an 866 bp spacer region (NTC8385-Min;contains P_(min) minimal pUC origin-RNA-OUT). These vectors were testedfor expression in vitro (lipofectamine 2000 delivery) and in vivo afterintradermal or intramuscular electroporation delivery. As withIntramuscular injection (Example 2, Table 1), the results withintradermal delivery (Table 10) demonstrated ColE2 and R6K originvectors dramatically improved in vivo expression compared to NTC8685.For example transgene expression from the NTC9385C vector wasunexpectedly improved 2.7 to 3.1× on days 4, 7, and 14 compared toNTC8685 after intradermal delivery (Table 10) and improved by 1.5 to3.8× on days 1, 4, 7, 14, 28 and 56 after intramuscular delivery.Transgene expression from the NTC9385R vector was unexpectedly improved5.3 to 6.3× on days 4, 7, and 14 compared to NTC8685 after intradermaldelivery (Table 10) and improved by 1.5 to 2.3× on days 1, 4, 7, 14, 28and 56 after intramuscular delivery. The 866 bp minimal pUC originvector also improved transgene expression to 1.4-1.9× that of NTC8685after intradermal delivery. This demonstrates improved in vivoexpression with the NTC9385C and NTC9385R vectors is not tissue specificsince expression improvement was obtained after intradermal andintramuscular delivery. Additionally, improved in vivo expression of theinvention is not specific to the CMV promoter since improved transgeneexpression was also observed with an NTC9385C-muSEAP vector with themurine creatine kinase (MCK) promoter substituted for the CMV promoter(NTC9385C-MCK-muSEAP, see Example 2). NTC9385C-MCK-muSEAP expression wasimproved 4.5× compared to NTC8685-MCK-muSEAP on day 28 afterintramuscular delivery with EP (98.4±55.8 versus 22.0±10.9 pg/mL) (seeExample 2). Inclusion of the C2×4 eukaryotic transcription terminator inthe NTC9385C vector further improved in vivo expression to 2.9 to 4.1×compared to NTC8685 after intradermal delivery (Table 10). These resultscollectively demonstrate improved in vivo expression with Nanoplasmidvectors may be obtained in various tissues and with alternativeeukaryotic promoters or with alternative/additional sequences flankingthe spacer region encoded bacterial region.

Nanoplasmid vectors additionally encoding RNA-OUT in the HTLV-1R Rabbitβ globin hybrid intron (both orientations of RNA-OUT SEQ ID NO:20inserted into the unique HpaI site in the intron (SEQ ID NO:1)(NTC9385Ra-O1 dual and NTC9385Ra-O2 dual) were constructed. Robustexpression with RNA-OUT in either orientation in the intron was observed(Table 8). The spacer region RNA-OUT was excised from these vectors(KpnI and DraIII digestion to excise RNA-OUT, ends blunted by T4 DNApolymerase treatment, blunt end ligation), to create NTC9385Ra-O1 (SEQID NO:50) and NTC9385Ra-O2 (SEQ ID NO:51) which have oppositeorientations of intronic RNA-OUT marker and only the R6K replicationorigin in the spacer region (SR=306 bp). Similarly high level expressionwith both clones was observed (Table 8). To determine splicing accuracyNTC9385Ra-O2-EGFP was transfected into the A549 cell line andcytoplasmic RNA isolated and splice junctions characterized as describedin Example 4. The RNA was reverse transcribed using an EGFP specificprimer, and PCR amplified using Exon 1 and Exon 2 specific primers. Theresultant PCR product (a single band) was determined by sequencing to bethe correct spliced exon1-exon2 fragment. This demonstrated thatintronic RNA-OUT is accurately removed by splicing and does notinterfere with splicing accuracy. NTC9385Ra-O2-EGFP also demonstratedimproved in vivo expression compared to NTC8685 (Table 10: 1.6-3.5×).Additionally, high yield manufacture was obtained with NTC9385Ra vectors(Table 7). This demonstrates that Nanoplasmid vectors with improvedtransgene expression of the current invention may encode the RNAselectable marker in the intron rather than the spacer region.

TABLE 10 SR vector expression in vitro and in vivo ID + EP ^(c) ID + EP^(c) ID + EP ^(c) muSEAP SR A549 HEK-293 (pg/mL) (pg/mL) (pg/mL) Vector^(b) SR ^(a) (bp) Intron ^(a) (A₄₀₅) ^(d) (A₄₀₅) ^(d) T = 4 T = 7 T = 14NTC8685 T-VA1- 1465 HR-β 0.240 ± 0.029 3.002 ± 0.188 1.9 ± 1.2 6.7 ± 4.15.0 ± 3.9 BH-P- (1.0×) (1.0×) (1.0×) (1.0×) (1.0×) AF→ NTC8385-T-P_(min)- 866 HR-β 0.495 ± 0.027 2.713 ± 0.177 3.7 ± 2.7 12.4 ± 8.1 7.1 ± 5.2 Min ^(e) AF→ (2.1×) (0.9×) (1.9×) (1.9×) (1.4×) NTC9385R T ←R- 466 HR-β 0.604 ± 0.04  3.036 ± 0.169 12.0 ± 7.4  35.5 ± 31.1 29.9 ±23.4 AF→ (2.5×) (1.0×) (6.3×) (5.3×) (6.0×) NTC9385C ←C- 281 HR-β 0.267± 0.053 2.720 ± 0.228 5.8 ± 3.0 20.8 ± 9.6  13.5 ± 9.8  AF→ (1.1×)(0.9×) (3.1×) (3.1×) (2.7×) NTC9385C ←C- 281 HR-β 0.214 ± 0.017 2.472 ±0.197 5.6 ± 2.3 27.7 ± 20.3 16.0 ± 14.3 C2x4 AF→ (0.89×) (0.82×) (2.9×)(4.1×) (3.2×) NTC9385R T ←R 306 HR- ← 0.524 ± 0.071 3.065 ± 0.220 3.6 ±2.8 23.4 ± 16.5 7.8 ± 8.0 a-O2 AF-β (2.2×) (1.0×) (1.9×) (3.5×) (1.6×)^(a) Prokaryotic terminator = T; HTLV-IR = HR; B globin 3′ acceptor site= β; RNA-OUT = AF; pUC origin = P; minimal pUC origin = P_(min); R6Kγorigin = R; ColE2-P9 origin = C; C2x4 eukaryotic transcriptionterminator = C2x4; PAS-BH = BH ^(b) All plasmids produced in XL1Bluedcm- host strains. P vectors were produced in dcm- XL1Blue NTC54208; Rvectors were produced in dcm- R6K Rep cell line NTC711231 (OL1 G to T);C vectors were produced in dcm- ColE2 Rep cell line NTC710351 (OL1 G toT). ^(c) Dose = 50 μg in 50 μl saline injected intradermal (ID) with EPon day 0. 6 mice/group. Mean ± SD pg/mL muSEAP reported for day 4, 7 and14. ( ) Mean muSEAP standardized to NTC8685 ^(d) muSEAP plasmid DNAtransfected with Lipofectamine 2000. Mean ± SD A₄₀₅ reported at 48 hrspost transfection. ( ) Mean A₄₀₅ standardized to NTC8685 ^(e) P_(min)minimal pUC origin (SEQ ID NO: 45) and RNA-OUT (bacterial region = SEQID NO:46)

The improved transgene expression level after intradermal deliverydemonstrates the application of Nanoplasmid and replicative minicirclevectors of the invention for cutaneous DNA vaccination and gene therapyapplications. For example, for intradermal DNA vaccination, epidermalDNA vaccination, or transcutaneous DNA vaccination using a variety ofantigens. For example, for gene therapy applications such as woundhealing, burns, diabetic foot ulcer, critical limb ischemia therapies,or cosmetic treatment for different dermatological conditions, includinganti-aging (anti-wrinkle), scar revision, radiation induced lesions,hair growth, surgical skin graft enhancement, or hemangioma, usinggrowth factors such as hypoxia inducible factor, hypoxia induciblefactor 1α, keratinocyte growth factor, vascular endothelial growthfactor (VEGF), fibroblast growth factor-1 (FGF-1, or acidic FGF), FGF-2(also known as basic FGF), FGF-4, placental growth factor (P1GF),angiotensin-1 (Ang-1), hepatic growth factor (HGF), DevelopmentallyRegulated Endothelial Locus (Del-1), stromal cell derived factor-1(SDF-1), etc.

Example 9: Alternative RNA Selectable Marker Nanoplasmid Vectors

The RNAI transcription unit (FIG. 10; SEQ ID NO: 34) was demonstrated inExample 7 as an acceptable substitute for the RNA-OUT selectable marker(SEQ ID NO: 20) in any of the constructs described in Examples 2-11.This may utilize the example pMB1 RNAI or the highly related ColE1 RNAI.

Another preferred RNA based selectable marker, IncB plasmid RNAI (SEQ IDNO:35) encoded within a selectable marker (SEQ ID NO:36), is shown inFIG. 11B. The promoter and terminator sequences flanking the IncBplasmid RNAI may be substituted with the plurality of promoter andterminator sequences know in the art. A cell line for antibiotic-freesucrose selection of IncB RNAI expressing plasmid vectors may be createdby modification of the genomically expressed RNA-IN-SacB cell lines forRNA-OUT plasmid propagation disclosed in Williams, Supra, 2008 byreplacement of the 68 bp RNA-IN regulator in a PstI-MamI restrictionfragment with a 362 bp PstI-MamI IncB RNAII regulator (SEQ ID NO:37)(FIG. 11A).

Another preferred RNA based selectable marker, an engineered CpG freerepressor RNA (SEQ ID NO: 38) encoded as part of a selectable marker(SEQ ID NO: 39), is shown in FIG. 12A. The promoter and terminatorsequences flanking the CpG free repressor RNA may be substituted withthe plurality of promoter and terminator sequences know in the art. ThisRSM represses a target RNA such as SEQ ID NO: 40 encoded upstream of atarget gene to be regulated such as SacB in SEQ ID NO: 41 and SEQ IDNO:42 (FIG. 12B; RNAS). pINT-RNAS expression vectors encoding SEQ IDNO:41 (P5/6 4/6 promoter driven expression of RNAS) and SEQ ID NO:42(P5/6 5/6 promoter driven expression of RNAS) RNAS PstI-BamHI (FIG. 12B)were constructed (SEQ ID NO: 43, SEQ ID NO: 44) as well as pINT-RNASvectors with a P5/6 6/6 promoter driving expression of RNAS. The P5/64/6, P5/6 5/6, P5/6 6/6 promoters, the pCAH63-CAT integration vector,and cloning and integration of pCAH63-CAT vector derivatives were asdescribed (Luke J, Carnes A E, Hodgson C P, Williams J A. 2009 Vaccine27:6454-6459). The P5/6 6/6 promoter contains a TAGACA-35 region that is5/6 match with the TTGACA-35 consensus sequence, separated, by theoptimal 17 bp spacing, from a TATAAT consensus-10 region. Briefly, thepCAH63-CAT integration vector was digested with BamHI and PstI andligated with a P5/6 6/6 promoter RNA-selection-SacB (RNAS) syntheticgene (Genscript, Piscataway, N.J.) which was excised as a 2487 bpPstI-BamHI restriction fragment. Clones (pINT-RNAS integration vectorP5/6 6/6) were verified by restriction digestion and integrated intoNTC54208 (XL1Blue dcm-) as described (Luke et al., Supra, 2009). Theresultant cell line, NTC781953 was demonstrated to be sucrose sensitiveas predicted. The 147 bp RNA-OUT selectable marker in NTC8685-EGFP wasexcised with DraIII/KpnI and the CpG free RNA selectable markersynthetic gene (Genscript, Piscataway, N.J.) excised as a 147 bpDraIII/KpnI restriction fragment (DraIII-SEQ ID NO: 39-KpnI) and ligatedto the 3672 bp NTC8685-EGFP DraIII/KpnI restriction fragment. Clones(NTC8685-RSM-EGFP) were identified as sucrose resistant colonies aftertransformation into NTC781953 and sequence validated. This demonstratesthat this alternative designed RNA selectable marker (SEQ ID NO: 38) maybe substituted for the RNA-OUT RNA selectable marker in the vectors ofthe current invention. RSM plasmids may be selected in alternative celllines, for example, in which the 6/6 consensus TATAAT-10 promoter regionin pINT-RNAS integration vector P5/6 6/6 was altered using syntheticoligonucleotides to 5/6 consensus TATGAT (P5/6 5/6) (pINT-RNAS P5/6 5/6;SEQ ID NO: 44) or 4/6 consensus TAGATT (P5/6 4/6) (pINT-RNAS P5/6 4/6;SEQ ID NO: 43). Alternatively, optimal promoter strength can bedetermined by other alterations in the −10 region that change theconsensus sequence, or alternations in the −35 region TTGACA consensus,or changes in the spacing between the −10 and −35 regions from theoptimal 17 bp. Engineered CpG free repressor RNA selectable markerreplicative minicircle vectors may be grown in RNA-selection-SacB (RNAS)regulated cell lines further expressing, as required, R6K, ColE2-P9, orColE2 related Rep protein. RNA-selection-SacB (RNAS) regulated celllines may also be made replacing the RNA-IN sequence in pCAH63-CATRNA-IN-SacB (P5/6 6/6) with the target RNA SEQ ID NO: 40 as described inWilliams, Supra, 2008 included herein by reference.

Example 10: Minimal pUC Origin Replicative Minicircles

Replicative minicircle vectors NTC8485P2 (0.85)-O1, NTC8485P2 (0.85)-O2,NTC9385P2a(0.85)-O1 and NTC9385P2a(0.85)-O2 containing the P_(min) pUCreplication origin (SEQ ID NO: 45) and the RNA-OUT RNA selectable marker(0.85 kb Bacterial region=SEQ ID NO: 46) within the intron wereconstructed as described in Example 3, and characterized for expressionin HEK293 and A549 (Table 11) and splicing accuracy in A549 (Table 9) asdescribed in Example 4.

TABLE 11 Robust expression with P2-(0.85) replicative minicircles A549FU^(b) HEK293 FU^(b) Vector (T = 48 (T = 48 (EGFP) Spacer region ^(a)Intron ^(a) mean + SD) mean + SD) NTC8485 T BH ← P-AF→ HR-β 4311 ± 45840236 ± 1851 (SV40-BE) (1×) (1×) NTC8485C2-O1 T BH ← P-AF→ HR ← C AF →-β 5001 ± 2724 41334 ± 14098 (SV40-BE) (1.16×) (1.03×) NTC8485C2-O2 T BH← P-AF→ HR ← AF C → -β 2962 ± 495 28849 ± 2421 (SV40-BE) (0.69×) (0.72×)NTC8485R2-O1 T BH ← P-AF→ HR ← T-R AF → -β 2888 ± 180 29395 ± 1054(SV40-BE) (0.67×) (0.73×) NTC8485R2-O2 T BH ← P-AF→ HR ← AF R-T → -β3187 ± 851 33044 ± 3515 (SV40-BE) (0.74×) (0.82×) NTC8485P2-O1 T BH ←P-AF→ HR ← T-P-AF → -β 1143 ± 392 20775 ± 6777 (SV40-BE) (0.27×) (0.52×)NTC8485P2-O2 T BH ← P-AF→ HR ← AF P-T → -β 1500 ± 169 16575 ± 2483(SV40-BE) (0.35×) (0.41×) NTC8485P2 T BH ← P-AF→ HR ← T-P_(min)-AF → -1969 ± 591 31883 ± 2750 (0.85)-O1 (SV40-BE) β (0.46×) (0.79×) NTC8485P2-T BH ← P-AF→ HR ← AF P_(min)-T → - 2171 ± 410 24733 ± 1417 (0.85)-O2(SV40-BE) β (0.50×) (0.61×) NTC9385P2a-O1 None (BE) HR ← T-P-AF → -β4445 ± 217 26181 ± 1643 (1.03×) (0.65×) NTC9385P2a-O2 None (BE) HR ← AFP -T →-β 3457 ± 426 23829 ± 1514 (0.80×) (0.59×) NTC9385P2a None (BE) HR← T-P_(min) -AF → - 6175 ± 258 38169 ± 2245 (0.85)-O1 β (1.43×) (0.95×)NTC9385P2a- None (BE) HR←AF P_(min) -T → - 6756 ± 583 35363 ± 3532(0.85)-O2 β (1.57×) (0.88×) NTC9385C2a-O1 None (BE) HR ← C AF → -β 5793± 820 36804 ± 6725 (1.34×) (0.91×) NTC9385R2a-O1 None (BE) HR ← T-R-AF →-β 7498 ± 859 37595 ± 5497 (1.74×) (0.93×) NTC9385R2a-O2 None (BE) HR ←AF R-T → -β 5815 ± 456 36926 ± 2001 (1.35×) (0.92×) ^(a) Prokaryoticterminator = T; HTLV-IR = HR; B globin 3′ acceptor site = β; RNA-OUT =AF; pUC origin = P; P_(min) minimalized pUC origin = P_(min); R6Kγorigin = R; ColE2-P9 origin = C; Boundary element = BE; 2 × 72 bp repeatof SV40 enhancer = SV40. Bracketed BE and or SV40 are spacer regionflanking eukaryotic sequences ^(b) EGFP plasmid DNA transfected withLipofectamine 2000. Mean ± SD Fluorescence units (FU) at 48 hrs posttransfection reported. ( ) Mean FU standardized to NTC8485

As with NTC9385P2a-O1 (Example 4) splicing was accurate withNTC9385P2a(0.85)-O1. Minor amounts of a cryptic P_(min) derived exonwere detected with NTC9385P2a(0.85)-O2; the sequence of the cryptic exonmatches the previously identified pUC derived cryptic exon observed withNTC9385P2a-O2, (Table 9; FIG. 3). Expression from both orientations wasunexpectedly higher with NTC9385P2a(0.85)-O1 and NTC9385P2a(0.85)-O2compared to NTC9385P2a-O1 and NTC9385P2a-O2 (Table 11) as well asNTC8485P2a(0.85)-O1 and NTC8485P2a(0.85)-O2 compared to NTC8485P2a-O1and NTC8485P2a-O2 (Table 11). While not limiting the application of thisinvention, the higher expression with the intronic P_(min) replicativeminicircles versus intronic pUC replicative minicircles may be due tosmaller intron size or deletion of inhibitory sequences, such as the pUCorigin nuclease sensitive site (FIG. 3). High yield manufacture wasobtained with these intronic P_(min) pUC replication origin vectors(Table 7) with high quality plasmid surprisingly without detectablereplication intermediates despite the close proximity of the P_(min) pUCreplication origin and the CMV promoter enhancer.

Example 11: 3′ UTR Nanoplasmid Vectors

The R6K origin (SEQ ID NO: 11), RNA-OUT selectable marker (SEQ ID NO:20), or R6K-RNA-OUT bacterial region (SEQ ID NO: 26) were cloned intothe 3′ UTR of the NTC7485 and NTC9385C vectors. NTC7485 is a kanamycinresistant (kanR) derivative of the NTC8485 vector in which RNA-OUT issubstituted with kanR NTC7485 was used to test expression of vectorswith the RNA-OUT selectable marker and R6K-RNA-OUT bacterial region inthe 3′ UTR to avoid duplication of RNA-OUT within a vector backbone.Likewise, NTC9385C was used to test expression with the R6K originencoded in the 3′ UTR since this vector does not encode the R6K origin.

NTC7485-EGFP-R-OUT O1 and O2 were constructed by cloning the RNA-OUTselectable marker as a 147 bp DraIII/KpnI (blunted with T4 DNApolymerase) restriction fragment into BglII digested (blunted by fillingwith Klenow), CIP treated NTC7485-EGFP (4508 bp restriction fragment).Recombinant clones of both orientations were identified as sucroseresistant colonies in cell line NTC54208 and confirmed by restrictionmapping and sequencing. The orientation 2 clone that was testedcontained two copies of RNA-OUT (Table 12). NTC7485-EGFP R6K-R-OUT O1and O2 were constructed by cloning the R6K-RNA-OUT bacterial region as a447 bp BsrBI/KpnI (blunted with T4 DNA polymerase) restriction fragmentinto BglII digested (blunted by filling with Klenow), CIP treatedNTC7485-EGFP (4508 bp restriction fragment). Recombinant clones of bothorientations were identified as sucrose resistant colonies in cell lineNTC54208 and confirmed by restriction mapping and sequencing.NTC9385C-EGFP-R6K O1 and 2 were constructed by cloning the R6K origin asa 300 bp BsrBI/DraIII (blunted with T4 DNA polymerase) restrictionfragment into BglII digested (blunted by filling with Klenow), CIPtreated NTC9385C-EGFP (2206 bp restriction fragment). Recombinant clonesof both orientations were identified as sucrose resistant colonies inR6K production cell line NTC711231 and confirmed by restriction mappingand sequencing.

Transgene (EGFP) expression of these 3′ UTR selection, replication orselection-replication vectors, compared to the parent vectors, wasdetermined in HEK293 and A549 cell lines as described in Example 4(Table 12). The observed robust expression of 3′UTR clones with RNA-OUT,R6K-RNA-OUT or R6K in either orientation demonstrated that 3′ UTRencoded replication and or selection is compatible with replicativeminicircle function and high level expression. Additionally, the resultsfurther demonstrate that 3′UTR selection can be combined with spacerregion replication (NTC7485-R-OUT O1, O2) or that 3′ UTR replication canbe combined with spacer region selection (NTC9385C-R6K-O1, O2).

The ←R-AF→, AF→, ←R orientations are preferred since these contain noopen reading frames which could be translated by read through of thetransgene stop codon (Table 12).

TABLE 12 High level expression with R6K replication origin and/orRNA-OUT encoded in the 3′ UTR Plasmid (all EGFP) Spacer ^(a) 3′ UTR^(a,) A549 EGFP ^(b) HEK EGFP ^(b) NTC7485 T-BH-P-kanR None ^(c) 3418 ±739 24066 ± 1169 (SV40-BE) (1×) (1×) NTC7485- T-BH-P-kanR ← R-AF → ^(c)2110 ± 233 23822 ± 2430 R6K-R-OUT O1 (SV40-BE) (0.61×) (0.99×) NTC7485-T-BH-P-kanR ← AF R → 1666 ± 228 18230 ± 823 R6K-R-OUT O2 (SV40-BE)(0.49×) (0.76×) NTC7485- T-BH-P-kanR AF→ ^(c) 2709 ± 332 25609 ± 1430R-OUT O1 (SV40-BE) (0.79×) (1.06×) NTC7485- T-BH-P-kanR ← AF ← AF 2151 ±207 19471 ± 1221 R-OUT O2 (2x) (SV40-BE) (0.63×) (0.81×) NTC9385C C-AF→None ^(c) 4044 ± 592 28546 ± 1370 (1.18×) (1.19×) NTC9385C- C-AF→ ←R^(c) 7897 ± 961 37645 ± 1264 R6K O1 (2.31×) (1.56×) NTC9385C- C-AF→ R→8305 ± 317 36707 ± 1024 R6K O2 (2.43×) (1.53) ^(a) Prokaryoticterminator = T; RNA-OUT = AF; pUC origin = P; R6Kγ origin = R; ColE2-P9origin = C; 2 × 72 bp repeat of SV40 enhancer = SV40; PAS-BH = BH.Bracketed BE or SV40 are spacer region flanking eukaryotic sequences^(b) EGFP plasmid DNA transfected with Lipofectamine 2000. Mean ± SDFluorescence units (FU) at 48 hrs post transfection reported. ( ) MeanFU standardized to NTC7485 ^(c) No open reading frames in 3′UTR (cutoffof minimum 20 amino acids)

A vector, NTC9385RbF (FIG. 13; SEQ ID NO: 47), that contains the R6Kminiorigin in the 3′UTR in the ←R (orientation 1) configuration (whichhas no 3′ UTR open reading frames), and intronic RNA-OUT was created asfollows. First, NTC9385C-Rbf-EGFP was constructed by cloning the R6Korigin as a 316 bp BfaI (klenow heat killed) then DraIII (blunted withT4 DNA polymerase) restriction fragment into BglII digested (blunted byfilling with Klenow), CIP treated NTC9385C-EGFP (2206 bp restrictionfragment). Recombinant clones of the correct orientation were identifiedas sucrose resistant colonies in R6K production cell line NTC711231 andconfirmed by restriction mapping and sequencing. This construct wasdigested with AlwNI and SacII to excise the intron, and the 2217 bprestriction fragment was ligated to the 449 bp RNA-OUT intron fromNTC9385Ra-O1-EGFP similarly digested with AlwNI and SacII. The resultantconstruct (NTC9385C-RbF-EGFP Intron RNA-OUT) was sequence verified thendigested with NotI and NcoI to excise the spacer region encoded ColE2origin-RNA-OUT. The resultant 2014 bp fragment was ligated to the spacerregion and boundary element from NTC9385R2a-O1-muSEAP as a compatible462 bp NotI and NcoI digested restriction fragment. The resultant clone,NTC9385RbF-EGFP was sequence validated, and surprisingly robustexpression (Table 13) and high fermentation yields (Table 7) verified.This demonstrates the surprising observation that replication andselection functions may be encoded within the 3′ UTR and intronrespectively. Collectively, these results demonstrate that 3′UTRselection can be combined with spacer region or intronic replication orthat 3′ UTR replication can be combined with spacer region or intronicselection. The RNA-OUT selectable marker may be substituted withalternative RNA selectable markers as described in Examples 7 and 9.

TABLE 13 High level expression with R6K replication origin encoded inthe 3′ UTR Vector (all EGFP) Spacer ^(a,b) Intron ^(a) 3′ UTR ^(a) A549EGFP ^(b) HEK EGFP ^(b) NTC9385C ← C-AF → HR-β None 2661 ± 489 15722 ±2235 (3.25×) (4.74×) NTC9385R T ← R AF → HR-β None 4803 ± 298 18396 ±2231 (5.86×) (5.55×) NTC8685 T-VA1-BH-P- HR-β None 2164 ± 364 18153 ±2251 AF (SV40) (2.64×) (5.47×) NTC9385R2a-O2 (BE) HR ← AF R → T-β None2967 ± 476 12581 ± 852 (3.62×) (3.79×) NTC9385R2a-O1 (BE) HR ← T-R-AF →-β None 3416 ± 283 15059 ± 2639 (4.17×) (4.54×) NTC9385Ra-O1 ←R HR-AF →-β None 2727 ± 315 19124 ± 4212 (3.33×) (5.77×) NTC9385RbF (BE) HR-AF →-β ←R 2427 ± 184 13257 ± 2720 (2.96×) (4.00×) pVAX1 P-kanR None None 820± 82 3317 ± 83 (1×) (1×) ^(a) trpA term = T; HTLV-IR = HR; B globin 3′acceptor site = β; RNA-OUT selectable marker = AF; pUC origin = P; R6Korigin = R; ColE2 origin = C; CMV boundary element = BE; PAS-BH = BH.Bracketed BE or SV40 are spacer region flanking eukaryotic sequences^(b) EGFP plasmid DNA transfected with Lipofectamine 2000. Mean ± SDFluorescence units (FU) at 48 hrs post transfection reported. ( ) MeanFU standardized to pVAX1

To demonstrate alternative RNA selectable markers can be substituted forRNA-OUT in the 3′ UTR, and that the ColE2 origin can be substituted forthe R6K origin in the 3′ UTR, NTC9385R-EGFP derivatives were made withthe RNAI selectable marker (SEQ ID NO: 34) or the ColE2 origin (+7) (SEQID NO: 13)-CpG free ssiA (SEQ ID NO: 16) inserted in the 3′ UTR. ControlNTC9385R-EGFP constructs with the pUC origin or the P_(min) minimalizedpUC origin (SEQ ID NO: 45) inserted in the 3′ UTR were also constructedand expression tested. NTC9585R was used to test expression of vectorswith the RNAI selectable marker and ColE2 and pUC replication origins inthe 3′ UTR to avoid duplication of RNAI, ColE2 or pUC sequences within avector backbone. All RNAI, ColE2 or pUC sequences were cloned as bluntended restriction fragments into the 3′ UTR of the NTC9385R-EGFP vectorthat had been digested with BglII, blunted by filling with klenow (2391bp restriction fragment), and CIP treated. RNAI selectable marker (SEQID NO: 34) was excised with HpaI as a 162 bp restriction fragment from asynthetic gene (Genscript, Piscataway, N.J.). The ColE2 origin (+7) (SEQID NO: 13)-CpG free ssiA (SEQ ID NO: 16) was excised from NTC9385C-EGFPas a 132 bp NheI (heat kill, klenow filled to blunt)/DraIII (heat kill,T4 DNA polymerase treatment to remove protruding sticky end) restrictionfragment. The pUC origin was excised from NTC8385-EGFP as an 1067 bpNheI (heat kill, klenow filled to blunt)/DraIII (heat kill, T4 DNApolymerase treatment to remove protruding sticky end) restrictionfragment. The P_(min) minimalized pUC origin was excised fromNTC8385-EGFP as an 720 bp AflIII/BspHI (heat kill, klenow filled toblunt) restriction fragment. Recombinant clones were identified assucrose resistant colonies in R6K replication cell line NTC711231 andconfirmed by restriction mapping and sequencing. Transgene (EGFP)expression of these 3′ UTR selection or replication or vectors, comparedto the parent NTC9385R-EGFP vector, was determined in HEK293 and A549cell lines. The results demonstrated robust expression with constructswith 3′ UTR encoded RNAI selectable marker or ColE2 origin-ssiAreplication origin but not with constructs encoded the pUC origin orP_(min) minimalized pUC origin (Table 14). This demonstrates that robustexpression replicative minicircle vectors can be constructed with theColE2 or R6K origin, and/or RNA selectable markers encoded in the 3′UTR.

The ←R-AF→, AF→←R, AF→, ←R, ←R-RNAI→, RNAI→←R, ←R←RNAI, ←RNAI←R, RNAI→,and ←RNAI compositions and orientations are preferred in the 3′ UTRsince these contain no open reading frames which could be translated byread through of the transgene stop codon (Table 14). RNA-OUT selectablemarker (SEQ ID NO: 20), CpG free RNA-OUT selectable marker (SEQ ID NO:22) and RSM (SEQ ID NO: 39) are preferred RNA selectable markers in the3′ UTR in the AF→ orientation since these RNA selectable markers containno open reading frames which could be translated by read through of thetransgene stop codon. The RNAI selectable marker (SEQ ID NO: 34) ispreferred in either orientation since both orientations do not containopen reading frames which could be translated by read through of thetransgene stop codon.

TABLE 14 High level expression with RNAI encoded in the 3′ UTR Vector(all EGFP) Spacer ^(a, b) Intron ^(a) 3′ UTR ^(a) A549 EGFP ^(b) HEKEGFP ^(b) NTC8685 T-VA1-BH- HR-β None ^(c) 5519 ± 483 51594 ± 1019 P-AF(SV40) (2.76×) (8.36×) NTC8685-RSM T-VA1-BH- HR-β None ^(c) 5655 ± 51248511 ± 4272 P-RSM(SV40) (2.83×) (7.86×) NTC9385R-Intron T ← R-AF →CMV-β None ^(c) 12361 ± 742 39832 ± 1273 (6.18×) (6.45×) NTC9385R T ←R-AF → HR-β None ^(c) 12036 ± 2401 50208 ± 1084 (6.01×) (8.14×)NTC9385R- T ← R-AF → HR-β P→ 3470 ± 362 9827 ± 595 3′UTR pUC O2 (1.73×)(1.59×) NTC9385R- T ← R-AF → HR-β ←P_(min) 2950 ± 130 10828 ± 715 3′UTRpMIN O1 (1.47×) (1.75×) NTC9385R- T ← R-AF → HR-β P_(min)→ 2010 ± 886523 ± 476 3′UTR pMIN O2 (1.00×) (1.06×) NTC9385R- T ← R-AF → HR-β ←C9569 ± 682 40691 ± 1421 3′UTR C2 O1 (4.78×) (6.59×) NTC9385R- T ← R-AF →HR-β ←RNAI ^(c) 9064 ± 295 35543 ± 2829 3′UTR RNAI O2 (4.53×) (5.76×)NTC9385RbF (BE) HR-AF → -β ←R ^(c) 8500 ± 1618 39407 ± 4006 (4.23×)(6.39×) pVAX1 P-kanR None None 2001 ± 299 6170 ± 778 (1×) (1×) ^(a) trpAterm = T; HTLV-IR = HR; B globin 3′ acceptor site = β; RNA-OUTselectable marker = AF; pUC origin RNAI antisense RNA selectable marker= RNAI; RSM antisense repressor RNA marker = RSM; pUC origin = P; pMINorigin = P_(min); R6K origin = R; ColE2 origin = C; CMV boundary element= BE; PAS-BH = BH; CMV B globin 3′ acceptor site = CMV- β = SEQ ID NO:10. Bracketed BE or SV40 are spacer region flanking eukaryotic sequences^(b) EGFP plasmid DNA transfected with Lipofectamine 2000. Mean ± SDFluorescence units (FU) at 48 hrs post transfection reported. ( ) MeanFU standardized to pVAX1 ^(c) No open reading frames in 3′UTR (cutoff ofminimum 20 amino acids)Summary

While the above description contains many examples, these should not beconstrued as limitations on the scope of the invention, but rathershould be viewed as an exemplification of preferred embodiments thereof.Many other variations are possible. For example, a replication originand/or a selectable marker may be inserted into the 3′ UTR at any sitebetween the transgene stop codon and the polyadenylation signal. Thepolyadenylation signal may be from a variety of polyadenylation signalsknown in the art, including the rabbit β globin, the human β globin,SV40 early, SV40 late, bovine growth hormone, etc, polyadenylationsignals. Additionally, a replication origin and/or a selectable markermay be inserted into the HTLV-I R-Rabbit β globin hybrid intron (SEQ IDNO:1) at any site between the 5′ splice acceptor and the 3′ acceptorbranch site (FIG. 1) rather than the HpaI site. Alternatively, areplication origin and/or a selectable marker may be inserted at twodifferent sites within an intron between the 5′ splice acceptor and the3′ acceptor branch site. Alternatively, a replication origin and aselectable marker may be inserted into two different introns, eachinsertion at any site between the 5′ splice acceptor and the 3′ acceptorbranch site. Alternatively, a replication origin and a selectable markermay be inserted into alternative introns at any site between the 5′splice acceptor and the 3′ acceptor branch site. A non limiting list ofalternative introns for insertion of a bacterial region to create anintron encoded bacterial region of the invention are SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:32. Replacement of theHTLV-IR-rabbit β globin 3′ acceptor site intron (SEQ ID NO: 1) with theCMV-rabbit β globin 3′ acceptor site intron (SEQ ID NO:10) in theNTC9385R vector (NTC9385R-intron; SEQ ID NO: 64) resulted in high levelexpression, comparable with the HTLV-IR-rabbit β globin 3′ acceptor siteintron containing NTC9385R vector (Table 14). This demonstrates thatvarious introns can be utilized to practice the invention.

Additionally, the RNA-OUT selectable marker may be substituted with analternative RNA-OUT sequence variant that functionally binds RNA-IN torepress expression. Likewise, the RNA-OUT promoter and/or terminatorcould be substituted with an alternative promoter and/or terminator.Further, an alternative RNA based selectable marker could be substitutedfor RNA-OUT. This may be a plasmid borne nonsense suppressing tRNA thatregulates a nonsense suppressible selectable chromosomal target asdescribed by Crouzet and Soubrier, Supra, 2005 included herein byreference. This may also be a plasmid borne antisense repressor RNA, anon limiting list included herein by reference includes pMB1 plasmidorigin encoded RNAI (SEQ ID NO: 33) that represses RNAII regulatedtargets (as described in Grabherr and Pfaffenzeller, Supra, 2006;Cranenburgh, Supra, 2009), plasmid pMU720 origin encoded RNAI (SEQ IDNO: 35) that represses RNA II regulated targets (Wilson et al., Supra,1997) ParB locus Sok of plasmid R1 that represses Hok regulated targets,Flm locus FlmB of F plasmid that represses flmA regulated targets(Morsey, Supra, 1999) or other antisense repressor RNAs known in theart.

For example, the pMB1 plasmid origin encoded RNAI (SEQ ID NO: 33) as aselectable marker (SEQ ID NO:34) flanked by restriction sites forcloning purposes can substituted for RNA-OUT in any of the vectorsdisclosed in Examples 2-11. For example, NTC9385RbF (FIG. 13; SEQ ID NO:47) substituted with the RNAI selectable marker (SEQ ID NO: 34) is shownas SEQ ID NO:49. NTC9385Ra-O1 (SEQ ID NO:50) or NTC9385Ra-O2 (SEQ IDNO:51) substituted with the RNAI selectable marker (SEQ ID NO: 34) areshown as SEQ ID NO:54 and SEQ ID NO:55 respectively. NTC9385RaF (SEQ IDNO: 56) in which the R6K origin is positioned in the spacer region andthe RNA-OUT selectable marker is positioned in the 3′ UTR, substitutedwith the RNAI selectable marker (SEQ ID NO: 34) is shown as SEQ IDNO:58.

RNAI (SEQ ID NO: 33) expressing vectors can be selected in cell linesthat encode RNAII regulated targets as described in Grabherr andPfaffenzeller, Supra, 2006; Cranenburgh, Supra, 2009. In these celllines, binding of RNAI to RNAII target sequences inserted in an mRNAupstream of the target gene start codon represses expression of thetarget gene. The target gene can encode a repressor protein, that itselfsuppresses expression of a second gene. In this manner, RNAI repressorRNA repression of the RNAII regulated target gene leads to expression ofthe second gene. If the second gene is essential for growth, thenplasmid containing cells can be selected under conditions wherein secondgene expression is required for growth. Alternatively, the target genecan encode a selectable conditionally toxic molecule, such as SacB. Inthis manner, RNAI repressor RNA mediated repression of the RNAIIregulated target gene leads to repression of toxin gene expressionallowing selection of plasmid containing cells under conditions whereintoxin gene expression eliminates cells without plasmid. The RNAII thatis used to regulate target gene expression can be the entire anti-RNAI(1-108) region, or be a RNAII fragment that contains the three loopRNAII region complementary to RNAI, for example anti-RNAI (10-108) (SEQID NO: 59) or be one or two RNAII loops complementary to RNAI asdisclosed in Grabherr and Pfaffenzeller, Supra, 2006. A non limitinglist of configurations of these RNAII molecules that can be used toregulate target gene expression are: 1) A RNAII-target gene fusion, inwhich the RNAII is positioned downstream of a ribosome binding site andATG start codon, and is in frame with the target gene start codon, suchthat the RNAII is translated in frame as an N terminal extension of thetarget protein as described in Grabherr and Pfaffenzeller, Supra, 2006.Plasmid borne RNAI binds expressed RNAII inhibiting translation of thefusion protein; 2) A RNAII-target gene dual cistron, in which the RNAIIis positioned downstream of a ribosome binding site and ATG start codonand upstream of an in frame stop codon and second ribosome binding sitewhich is upstream of the target gene start codon, such that the RNAII istranslated as a first cistron, followed by translation of the targetprotein in a second cistron. Plasmid borne RNAI binds expressed RNAIIinhibiting translation of the first cistron, which prevents ribosomebinding to the second ribosome binding site, reducing target geneexpression; 3) RNAII leader upstream of target gene, in which the RNAIIis positioned upstream or overlapping the ribosome binding site of thetarget gene such that plasmid borne RNAI binds expressed RNAII RNApreventing ribosome binding to the ribosome binding site, reducingtarget gene expression. Configurations for RNAII leaders using ananti-RNAI (4-108) are disclosed in Cranenburgh, Supra, 2009. AlternativeRNAII leaders using an anti-RNAI(10-108) (SEQ ID NO: 59) with a weakribosome binding site (TCGA) upstream of the target gene ATG or a strongribosome binding site (AGGAGA) upstream of the target gene ATG are shownas SEQ ID NO:60 and SEQ ID NO:61 respectively. These cassettes can beexpressed from a variety of promoters, for example the P5/6 6/6, P5/65/6, or P5/6 4/6 promoters disclosed herein, regulate a variety oftarget genes, for example SacB or tetR disclosed herein, and integratedinto the genome using PCR products or integration vectors, for examplethe pINT integration vector disclosed herein.

Alternatively, an engineered RNA selectable marker such as the RSMantisense repressor RNA (SEQ ID NO: 38) may be substituted for RNA-OUT.The RSM antisense repressor RNA selectable marker (SEQ ID NO: 39) may beflanked by DraIII and KpnI restriction sites to allow precisereplacement of the RNA-OUT selectable marker (SEQ ID NO: 20) flanked byDraIII and KpnI sites. For example, the RNA-OUT marker was replaced withthe RSM antisense RNA marker (SEQ ID NO: 39) in NTC8685-RSM-EGFP (seeExample 9). The resultant vector had high expression in A549 and HEK293cells comparable to the RNA-OUT comparator (Table 14) demonstrating thatalternative RNA selectable markers can be utilized in the practice ofthe current invention. NTC9385RbF (FIG. 13; SEQ ID NO: 47) substitutedwith the RSM antisense repressor RNA marker (SEQ ID NO: 39) is shown asSEQ ID NO:48. NTC9385Ra-O1 (SEQ ID NO:50) or NTC9385Ra-O2 (SEQ ID NO:51)substituted with the RSM antisense repressor RNA marker (SEQ ID NO: 39)are shown as SEQ ID NO:52 and SEQ ID NO:53 respectively. NTC9385RaF (SEQID NO: 56) in which the R6K origin is positioned in the spacer regionand the RNA-OUT selectable marker is positioned in the 3′ UTR,substituted with the RSM antisense repressor RNA marker (SEQ ID NO: 39)is shown as SEQ ID NO:57.

For CpG free vector applications, the CpG free RNA-OUT selectable marker(SEQ ID NO: 22) or RSM antisense repressor RNA marker (SEQ ID NO: 39)may be flanked by CpG free restriction enzyme sites, for example BglIIor EcoRI for cloning, or may be incorporated into the vector by PCR, orby synthesizing the new vector de novo using gene synthesis. The CpGfree RNA selectable marker may be incorporated within an intron, a 3′UTR or the spacer region of a vector. CpG free replication origins maybe incorporated within an intron, a 3′ UTR or the spacer region of avector. The CpG free RNA selectable markers and replication origins maybe incorporated together or separately with introns, a 3′ UTR or thespacer region of a vector. A CpG free RNA selectable marker may becombined with a CpG free R6K replication origin (e.g. SEQ ID NO: 12) inany orientation to make a CpG free bacterial region, for example SEQ IDNO: 28. A CpG free RNA selectable marker may be combined with a CpG freeColE2 replication origin (e.g. SEQ ID NO: 16) in any orientation,optionally incorporating a CpG free ssi (e.g. SEQ ID NO: 17), to make aCpG free bacterial region, for example SEQ ID NO: 25. These CpG freebacterial regions may be incorporated into the spacer region, the intronor the 3′ UTR of a vector.

In the vectors of the invention, the ColE2-P9 or R6K replication originmay be substituted with a ColE2 related replication origin, andpropagated in a strain expressing the ColE2 related replication originreplication protein. Likewise, the ColE2-P9 or R6K Rep protein dependentorigin may be substituted with an origin from one of the numerousalternative Rep protein dependent plasmids that are know in the art, forexample the Rep protein dependent plasmids described in del Solar etal., Supra, 1998 which is included herein by reference. Likewise, thevarious orientations of the replication origin, and the RNA selectablemarker, may be utilized. For example, Table 15 summarizes the eightorientations of the replication origin, and the RNA selectable marker invectors of the current invention in which the replication origin and RNAselectable marker are both encoded together within either the spacerregion, a intron, or the 3′ UTR. Table 16 summarizes twenty fourorientations of the replication origin, and the RNA selectable marker invectors of the current invention in which the replication origin and RNAselectable marker are encoded separately within the spacer region, aintron, or the 3′ UTR. Vectors in which the replication origin and RNAselectable marker are encoded separately within the spacer region andthe 3′ UTR do not need to include an intron. However, one or moreintrons may optionally be included in vectors in which the replicationorigin and RNA selectable marker are encoded separately within thespacer region and the 3′ UTR.

TABLE 15 Spacer region, intron or 3′ UTR encoded RSMselection/replication origin short spacer region replicative minicirclevector configurations Vector Intron Vector Spacer region Vector 3′ UTR #configurations ^(a,b) configurations ^(a,c) configurations ^(a,c) 1 SD ←Rep RSM → SA PA ← Rep RSM → EP Stop ← Rep RSM → PA 2 SD ← Rep ← RSM SAPA ← Rep ← RSM EP Stop ← Rep ← RSM PA 3 SD Rep → RSM → SA PA Rep → RSM →EP Stop Rep → RSM → PA 4 SD Rep → ← RSM SA PA Rep → ← RSM EP Stop Rep →← RSM PA 5 SD ← RSM Rep → SA PA ← RSM Rep → EP Stop ← RSM Rep → PA 6 SD← RSM ← Rep SA PA ←RSM ← Rep EP Stop ← RSM ← Rep PA 7 SD RSM → Rep → SAPA RSM → Rep → EP Stop RSM → Rep → PA 8 SD RSM → ← Rep SA PA RSM → ← RepEP Stop RSM → ← Rep PA ^(a) SD = Splice donor; SA = Splice acceptor; Rep= replication origin, selected from the group R6K gamma replicationorigin, a ColE2-P9 replication origin, a ColE2-P9 related replicationorigin, a pUC replication origin (intron only, not in SR or 3′ UTR), aP_(min) pUC replication origin (intron only, not in SR or 3′ UTR); RSM =RNA selectable marker; Stop = transgene stop codon; PA = polyadenylationsignal; EP = RNA polymerase I, II or III enhancer promoter ^(b)Additional functional groups may be encoded within the intron, includingbacterial transcriptional terminators, eukaryotic promoters, eukaryoticenhancers, eukaryotic intronic splicing enhancers, nuclear localizingsequences, supercoiling-induced DNA duplex destabilized (SIDD)structures, microRNAs and/or immunostimulatory RNA elements etc ^(c)Additional functional groups may be encoded within the spacer region or3′ UTR, including bacterial transcriptional terminators, eukaryotictranscriptional terminators, eukaryotic enhancers, boundary elements,nuclear localizing sequences, supercoiling-induced DNA duplexdestabilized (SIDD) structures, microRNAs, mRNA export sequences (3′UTR), and/or immunostimulatory RNA elements etc

TABLE 16 Spacer region, intron or 3′ UTR encoded separated RSMselection/replication origin short spacer region replicative minicirclevector configurations Vector Intron Vector Spacer region Vector 3′ UTR #configurations ^(a,b) configurations ^(a,c) configurations ^(a,c) 1 SD ←Rep SA PA RSM → EP Stop PA 2 SD ← Rep SA PA ← RSM EP Stop PA 3 SD ← RepSA PA EP Stop RSM → PA 4 SD ← Rep SA PA EP Stop ← RSM PA 5 SD Rep → SAPA RSM → EP Stop PA 6 SD Rep → SA PA ← RSM EP Stop PA 7 SD Rep → SA PAEP Stop RSM → PA 8 SD Rep → SA PA EP Stop ← RSM PA 9 SD ← RSM SA PA Rep→ EP Stop PA 10 SD ← RSM SA PA ← Rep EP Stop PA 11 SD ← RSM SA PA EPStop Rep → PA 12 SD ← RSM SA PA EP Stop ← Rep PA 13 SD RSM → SA PA Rep →EP Stop PA 14 SD RSM → SA PA ← Rep EP Stop PA 15 SD RSM → SA PA EP StopRep → PA 16 SD RSM → SA PA EP Stop ← Rep PA 17 SD SA PA Rep → EP StopRSM → PA 18 SD SA PA Rep → EP Stop ← RSM PA 19 SD SA PA ← Rep EP StopRSM → PA 20 SD SA PA ← Rep EP Stop ← RSM PA 21 SD SA PA RSM → EP StopRep → PA 22 SD SA PA RSM → EP Stop ← Rep PA 23 SD SA PA ← RSM EP StopRep → PA 24 SD SA PA ← RSM EP Stop ← Rep PA ^(a) SD = Splice donor; SA =Splice acceptor; Rep = replication origin, selected from the group R6Kgamma replication origin, a ColE2-P9 replication origin, a ColE2-P9related replication origin, a pUC replication origin (intron only, notin SR or 3′ UTR), a P_(min) pUC replication origin (intron only, not inSR or 3′ UTR); RSM = RNA selectable marker; Stop = transgene stop codon;PA = polyadenylation signal; EP = RNA polymerase I, II or III enhancerpromoter ^(b) Additional functional groups may be encoded within theintron, including bacterial transcriptional terminators, eukaryoticpromoters, eukaryotic enhancers, eukaryotic intronic splicing enhancers,nuclear localizing sequences, supercoiling-induced DNA duplexdestabilized (SIDD) structures, microRNAs and/or immunostimulatory RNAelements etc ^(c) Additional functional groups may be encoded within thespacer region or 3′ UTR, including bacterial transcriptionalterminators, eukaryotic transcriptional terminators, eukaryoticenhancers, mRNA export sequences (3′ UTR), boundary elements,supercoiling-induced DNA duplex destabilized (SIDD) structures, nuclearlocalizing sequences, microRNAs and/or immunostimulatory RNA elementsetc

The vectors may encode a diversity of transgenes different from theexamples provided herein, for example, antigen genes for a variety ofpathogens, or therapeutic genes such as hypoxia inducible factor,keratinocyte growth factor, factor IX, factor VIII, etc, or RNA genessuch as microRNAs or shRNA. Likewise, the vectors may utilize adiversity of RNA Pol II promoters different from the CMV promoterexamples provided herein, for example, constitutive promoters such asthe elongation factor 1 (EF1) promoter, the chicken β-actin promoter,the β-actin promoter from other species, the elongation factor-1α (EF1α)promoter, the phosphoglycerokinase (PGK) promoter, the Rous sarcomavirus (RSV) promoter, the human serum albumin (SA) promoter, the α-1antitrypsin (AAT) promoter, the thyroxine binding globulin (TBG)promoter, the cytochrome P450 2E1 (CYP2E1) promoter, etc. The vectorsmay also utilize combination promoters such as the chicken β-actin/CMVenhancer (CAG) promoter, the human or murine CMV-derived enhancerelements combined with the elongation factor 1α (EF1α) promoters, CpGfree versions of the human or murine CMV-derived enhancer elementscombined with the elongation factor 1α (EF1α) promoters, the albuminpromoter combined with an α-fetoprotein MERII enhancer, etc, or thediversity of tissue specific or inducible promoters know in the art suchas the muscle specific promoters muscle creatine kinase (MCK), and C5-12or the liver-specific promoter apolipoprotein A-I (ApoAI). Theorientation of the various vector-encoded elements may also be changedrelative to each other.

The vectors may optionally contain additional functionalities, such asnuclear localizing sequences, and/or immunostimulatory RNA elements asdisclosed in Williams, Supra, 2008 as part of the eukaryotic region oralternatively within introns or within the spacer region.

Additional sequences may be added to the spacer, for example aeukaryotic selectable marker, bacterial transcription terminators,eukaryotic transcription terminators, boundary elements,supercoiling-induced DNA duplex destabilized (SIDD) structures, S/MARs,RNA Pol I or RNA Pol III expressed sequences or other functionalities.For example, improved transgene expression was observed when the gastrineukaryotic transcription terminator was inserted into the spacer region(NTC9385R2a-O2-Gt versus NTC9385R2a-O2; Table 8). As well, additionalsequences could be encoded within the intron, such as RNA Pol IIItranscription units expressing short hairpin RNA's, microRNAs orimmunostimulatory RNAs such as those disclosed in Williams, Supra, 2008,included herein by reference.

Any eukaryotic expression vector can be converted into replicativeminicircle expression vector of the invention by: 1) Cloning a RNAselectable marker and/or replication origin into an intron, 3′ UTR, orspacer region; and 2) removing the existing vector spacer region encodedselection marker and/or replication origin. If the vector does notcontain an intron, an intron for insertion of the bacterial region canbe added by standard cloning methodologies known in the art. More thanone intron can be used to make a replicative minicircle, by cloning thereplication origin into one intron and the selectable marker into asecond intron. Alternatively, the replication origin can be cloned intoan intron or UTR, and the selection marker encoded within the spacerregion created from excision of the existing vector encoded bacterialregion. Cloning may be performed using restriction enzyme fragmentligation or ligation independent cloning, or the various PCRamplification based cloning strategies known in the art. Alternatively,the vectors of the invention can be created de novo using gene synthesisto make the entire vector or fragments of the vector.

Thus, the reader will see that the improved replicative minicircleexpression vectors of the invention provide for an approach to improveplasmid encoded transgene expression (i.e. through incorporation of ashort spacer region preferably less than 500 bp) while dramaticallyimproving manufacture compared to alternative short spacer regionvectors such as minicircles.

Accordingly, the scope of the invention should be determined not by theembodiments illustrated, but by the appended claims.

What is claimed is:
 1. A method of constructing a eukaryotic replicativeminicircle expression vector and expressing a gene of interest therefromcomprising: a) combining i) a eukaryotic region with 5′ and 3′ endsencoding a gene of interest and comprising an intron, the introncomprising a 5′ splice donor and a 3′ acceptor branch site, a bacterialreplication origin selected from the group consisting of R6K replicationorigin, and ColE2-P9 replication origin, and an RNA selectable marker;with ii) a spacer region linking the 5′ and 3′ ends of the eukaryoticregion, said spacer region being less than 500 basepairs in length, tocreate a eukaryotic replicative minicircle expression vector; and b)introducing said replicative minicircle expression vector into a targeteukaryotic cell or a eukaryotic organism comprising the targeteukaryotic cell, under conditions wherein the target eukaryotic cell istransfected and said gene of interest is expressed.
 2. The method ofclaim 1, wherein said RNA selectable marker is an RNA-IN regulatingRNA-OUT functional variant with at least 95% sequence identity to asequence selected from the group consisting of SEQ ID NO:20, and SEQ IDNO:22.
 3. The method of claim 1, wherein said RNA selectable marker isselected from the group consisting of: an RNA-OUT selectable marker thatencodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequenceidentity to SEQ ID NO: 21; an RNAI selectable marker that encodes anRNAII regulating RNAI RNA with at least 95% sequence identity to SEQ IDNO: 33; an IncB RNAI selectable marker encoding an RNAII regulating RNAIRNA with at least 95% sequence identity to SEQ ID NO: 35; and ansynthetic RNA selectable marker encoding an RNA selectable markercomplement regulating RNA with at least 95% sequence identity to SEQ IDNO:
 38. 4. The method of claim 1, wherein said intron with a 5′ splicedonor and a 3′ acceptor branch site is a functional variant with atleast 95% sequence identity to a sequence selected from the groupconsisting of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10.
 5. The method of claim 1, wherein said pUC replication origin andsaid RNA selectable marker positioned within said intron is a functionalvariant with at least 95% sequence identity to SEQ ID NO:
 29. 6. Amethod of constructing a eukaryotic replicative minicircle expressionvector comprising: a) combining i) a eukaryotic region with 5′ and 3′ends encoding a gene of interest and comprising an intron the introncomprising a 5′ splice donor and a 3′ acceptor branch site, a bacterialregion comprising an R6K origin or a ColE2-P9 bacterial replicationorigin, and a RNA selectable marker with ii) a spacer region linking the5′ and 3′ ends of the eukaryotic region sequences, said spacer regionbeing less than 500 basepairs in length, to create a eukaryoticreplicative minicircle expression vector; b) transforming saidreplicative minicircle expression vector into cells of an RNA selectablemarker regulated bacterial cell line; c) isolating the resultanttransformed bacterial cells by selection; and d) propagating theresultant transformed bacterial cells in culture.
 7. The method of claim6, wherein said RNA selectable marker is an RNA-IN regulating RNA-OUTfunctional variant with at least 95% sequence identity to a sequenceselected from the group consisting of SEQ ID NO:20, and SEQ ID NO:22. 8.The method of claim 6, wherein said RNA selectable marker is selectedfrom the group consisting of: an RNA-OUT selectable marker that encodesan RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity toSEQ ID NO: 21; an RNAI selectable marker that encodes an RNAIIregulating RNAI RNA with at least 95% sequence identity to SEQ ID NO:33; an IncB RNAI selectable marker encoding an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 35; and an syntheticRNA selectable marker encoding an RNA selectable marker complementregulating RNA with at least 95% sequence identity to SEQ ID NO:
 38. 9.The method of claim 6, wherein said intron with a 5′ splice donor and a3′ acceptor branch site is a functional variant with at least 95%sequence identity to a sequence selected from the group consisting of:SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10.
 10. Themethod of claim 6, wherein said pUC replication origin and said RNAselectable marker positioned within said intron is a functional variantwith at least 95% sequence identity to SEQ ID NO:
 29. 11. A eukaryoticreplicative minicircle expression vector comprising i) a eukaryoticregion sequence with 5′ and 3′ ends encoding a gene of interest andcomprising an intron, the intron comprising a 5′ splice donor and a 3′acceptor branch site, a bacterial replication origin selected from thegroup consisting of R6K replication origin, and ColE2-P9 replicationorigin, and an RNA selectable marker; and ii) a spacer region of lessthan 500 basepairs in length linking the 5′ and 3′ ends of theeukaryotic region sequences.
 12. The vector of claim 11, wherein saidRNA selectable marker is an RNA-IN regulating RNA-OUT functional variantwith at least 95% sequence identity to a sequence selected from thegroup consisting of SEQ ID NO:20, and SEQ ID NO:22.
 13. The vector ofclaim 11, wherein said RNA selectable marker is selected from the groupconsisting of: an RNA-OUT selectable marker that encodes an RNA-INregulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO:21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNAwith at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAIselectable marker encoding an RNAII regulating RNAI RNA with at least95% sequence identity to SEQ ID NO: 35; an synthetic RNA selectablemarker encoding an RNA selectable marker complement regulating RNA withat least 95% sequence identity to SEQ ID NO:
 38. 14. The vector of claim11, wherein said intron with a 5′ splice donor and a 3′ acceptor branchsite is a functional variant with at least 95% sequence identity to asequence selected from the group consisting of: SEQ ID NO: 1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10.
 15. The vector of claim 11,wherein said pUC replication origin and said RNA selectable markerpositioned within said intron is a functional variant with at least 95%sequence identity to SEQ ID NO: 29.